KR101130587B1 - Glucose transport mutants for production of biomaterial - Google Patents

Abstract

A method of restoring the Glu ⁺ phenotype to PTS ⁻ / Glu ⁻ bacterial cells that is originally available for phosphotransferase transport systems (PTS) for carbohydrate transport is disclosed. Bacterial cells comprising the Glu ⁺ phenotype have altered endogenous chromosomal regulatory regions operably linked to polynucleotides encoding galactose permease and glucokinase.

Description

GLUCOSE TRANSPORT MUTANTS FOR PRODUCTION OF BIOMATERIAL}

Cross Reference to Related Application

This application claims priority to US Provisional Application No. 60 / 416,166, filed Oct. 4, 2002 and US Provisional Application No. 60 / 374,931, filed Oct. 4, 2002, which is hereby incorporated by reference.

The present invention relates to genetically engineered metabolic pathways in bacterial host cells and provides methods and systems for the preparation of desired products in engineered host cells. In particular, the present invention relates to the enhancement of glucose transport in host strains, which reduces PTS phosphoenolpyruvate (PEP) consumption and converts PEP or PEP precursors into preferred metabolic pathways such as conventional aromatic amino acid pathways. By altering the direction of, the original phosphoenolpyruvate (PEP): phosphate transferase transport system (PTS) can be used for glucose transport.

Many industrially important microorganisms use glucose as their primary carbon source to produce biosynthetic products. Thus, cost-effective and efficient biosynthetic preparation of the product requires a carbon source such as glucose that is converted to the product in a high percentage yield. In order to meet this need, it may be advantageous to increase the influx of carbon sources through or through various metabolic pathways such as conventional aromatic pathways, TCA pathways, and analytic oxaloacetate synthesis pathways.

In the early stages of host cell carbohydrate metabolism, each glucose molecule is converted into two phosphoenolpyruvate (PEP) molecules in a cytosol. PEP is one of the major metabolic building blocks that cells use in their biosynthetic pathways. For example, PEP can be further converted to pyruvate, and the chemical reaction of converting glucose to pyruvate is referred to as the Embden-Meyerhoff pathway. All metabolic intermediates between the original glucose carbohydrate and the final product, pyruvate, are all phosphorylated compounds. Bacteria that ferment glucose through the Embden-Meyerhoff pathway, such as Enterobacteriacea and Vibrionaceae , are described in Bouvet et al. (1989) International Jounal of Systematic Bacteriology , 39: 61-67. Pyruvate is then metabolized to give products such as lactate, ethanol, formate, acetate and acetyl CoA (see FIGS. 1A and 1B).

In addition to the Embden-Meyerhoff pathway, many bacteria have an active transport system known as phosphoenolpyruvate (PEP) -dependent phosphatase transport system (PTS). The system is coupled to its phosphorylation to transport carbon sources such as glucose. The phosphate group is sequentially transferred from PEP to enzyme I and from enzyme I to protein Hpr. The actual translocation step is catalyzed by the family of membrane binding enzymes (called enzymes II), each of which is specific for one or more carbon sources. As a reference, Postma et al., (1993) Phosphoenolpyruvate: Carbohydrate Phosphotransferase Systems in Baceteria, Microbiol. Reviews. 57: 573-594 and Postma PW (1996) Phosphotransferase System for Glucose and Other Sugars. In: Neidhardt et al., Eds. Escherichia Coli and Salmonella Typhimurium: Cellular and Molecular Biology. Vol. 1. Washington, DC ASM Press pp 127-141. However, due to the fact that PTS metabolizes PEP to phosphorylate the carbon source, the PTS system reduces the conversion efficiency of the carbon substrate to the desired product. In glycolysis, two PEP molecules form all catabolized glucose molecules. However, one PEP molecule is needed for the PTS to function, leaving only one PEP molecule needed for another biosynthetic reaction.

Because of its role as the central matabolite of PEP, numerous approaches have been used to increase PEP supply in cells, some of which are:

a) elimination of pyruvate kinase activity by generation of pyk mutations. Pyruvate kinase catalyzes the conversion of PEP to pyruvate (Mori et al. , (1987) Agric. Biol. Chem. 51: 129-138);

b) elimination of PEP carboxylase activity by generation of ppc mutations. PEP carboxylase catalyzes the conversion of PEP to oxalo acetate (Miller et al., (1987) J. Ind. Microbiol. 2: 143-149);

c) Expression amplification of pps encoding PEP synthase. PEP synthase catalyzes the conversion of pyruvate to PEP (USSN 08 / 307,371); And

d) for example, overexpression of the transketolase gene ( tkt A or tkt B) (US Pat. No. 5,168,056) or overexpression of the transaldolase gene ( tal A) (Iida et al., (1983) J. Bacteriol. 175: 5375-5383) to increase the supply of D-erythros-4-phosphate (E4P). Transketolase catalyzes the conversion of D-fructose-6-phosphate to E4P, and transaldolase converts D-sedoheptulose-7-phosphate + E4P + fructose- of glyceraldehyde-3-phosphate Catalyzes the conversion to 6-phosphate.

In addition to the above approaches, the researchers looked for ways to reduce PTS: PEP dependent consumption by eliminating or altering the action of PTS. This approach is also attractive because PEP energizes twice as much as ATP. Many of these efforts focus on the use of inert PTS systems. Examples of studies dealing with PTS systems include:

a) recovery of the glucose phenotype (Glu ⁺ ) in PTS inactivated E. coli cells by introducing glf and glk genes encoding glucose-promoting diffusion proteins and glucokinases derived from Zymomonas mobilis , wherein E. coli cells Has PTS inactivated by deletion of pstHlcrr operon (US Pat. No. 5,602,030 and Snoep et al., (1994) J. Bact. 176: 2133-2135) and

b) PTS ^- / Glu ^- apply the E. coli strains in continuous culture on a glucose selection and W, the wild-type PTS ^- / Glu ^- the capacity to obtain equivalent or higher growth rates as strain Glu ⁺ return variant (revertant) ( obtained a / GL ⁺⁾ (Flores, ^{etc., (2002) Metab Eng 4 -} PTS:.. 124 - 137; Flores , etc., (1996) Nature Biotechnol 14: 620 - 623 and WO96 / 34961).

However, the above approaches have various limitations. In general, the use of heterologous genes does not always work efficiently in new hosts. In addition, membrane proteins, such as glucose-promoting diffusion proteins, are usually intimately associated with lipids in cell membranes, which can vary from species to species. Introduced soluble proteins, such as glucokinase, can be applied for protease degradation. In addition, the use of spontaneous mutations in cells to regain the phenotype can have unpredictable results, and it is desirable for industrial processes to use fully characterized strains.

In contrast to this method, the present invention increases the carbon flow to metabolic pathways within bacterial strains that can transport glucose without consuming PEPs during the process. The converted PEP or PEP precursor may then be redirected to a given metabolic pathway for enhanced production of the desired product. Endogenous chromosomal regulation operably linked to glucose assimilation proteins and more specifically to glucose transporters and / or glucose phosphorylated proteins, to restore or regain the ability of cells to use glucose as a carbon source while maintaining inactivated PTS. By modification of the region, the strains are generated in cells with inactivated PEP-dependent PTS. The cells are designated PTS ⁻ / Glu ⁺ .

Summary of the Invention

Thus, the present invention provides a method of increasing carbon flow into a metabolic pathway of a bacterial host cell, wherein the host cell may originally use PTS for carbohydrate transport. The method comprises the selection of a bacterial host cell whose phenotype is PTS ⁻ / Glu ⁻ and modification of an endogenous chromosomal regulatory region operably linked to a nucleic acid encoding a polypeptide involved in glucose assimilation to restore the Glu ⁺ phenotype. do.

In a first aspect, the present invention relates to a method of increasing carbon flow into the metabolic pathway of PTS ⁻ / Glu ⁻ bacterial host cells, which may originally use a phosphatase transport system (PTS) for carbohydrate transport, The method comprises the steps of: a) transforming PTS ⁻ / Glu ⁻ host cells using a DNA construct comprising a promoter and a contiguous DNA sequence corresponding to the upstream (5 ′) region of the glucose assimilation protein, thereby transforming the PTS ⁻ / Glu ^- changing an endogenous chromosome regulatory region operably linked to a nucleic acid encoding a glucose assimilation protein in a host cell; b) allowing the Glu ⁺ phenotype to be restored by incorporation of the DNA construct; And c) culturing the transformed host cell under appropriate culture conditions, wherein the carbon flow into the metabolic pathway of the transformed host cell is directed to the same metabolic pathway in the corresponding PTS bacterial host cell cultured under essentially the same culture conditions. Increased compared to the carbon flow of. In a first embodiment of the method, the promoter is a non-host cell promoter or a modified endogenous promoter. In a second embodiment, the glucose assimilation protein is a glucose transporter, preferably a galactose permease obtained from E. coli or a glucose transporter having at least 80% sequence homology with it. . In a third embodiment, the glucose assimilation protein is a glucokinase obtained from a phosphorylated protein, preferably E. coli or a glucokinase having at least 80% sequence homology with it. In a fourth embodiment of the method, is selected from the bacterial host cell is Escherichia coli (E. coli) cells, Bacillus (Bacillus) cell, and the group consisting of O (Patoea) cells in a panto. In the fifth embodiment, PTS ^- / Glu ^- host cells by deletion of one or more genes selected from the group consisting of l pts, pts H and crr, is obtained from the PTS cell. In the sixth embodiment, PTS ^- / Glu ⁺ host cells, trans Kane Tortola azepin trans aldolase, play pyruvate in phosphorylation bait synthase, DAHP synthase, DHQ synthase, DHQ di-hydrazide hydratase, to mate It is transformed using a polynucleotide encoding a protein selected from the group consisting of dehydrogenase, schimate kinase EPSP synthase and corismate synthase.

In a second aspect, the present invention is described in the first aspect, and using the second DNA construct comprising a contiguous DNA sequence homologous to the upstream (5 ') region of the promoter, and the glucokinase, PTS ^- / Glu ^- by transforming a host cell, PTS ^- / Glu ^- relates to a process comprising the transformation of a host is operably linked to a nucleic acid encoding the glucokinase, the endogenous chromosomes in the cells that control area.

In a third aspect, the invention features the original carbohydrate transport PTS available in the PTS for the ^- / Glu ^- comprises relates to a process for in the host bacterial cell, increase the production of the desired product, which steps of: a ) Transforming a bacterial host cell with a PTS ⁻ / Glu ⁻ phenotype with a DNA construct comprising a promoter, wherein said DNA construct replaces an endogenous promoter operably linked to a nucleic acid encoding a glucose assimilation protein. Integrated into the PTS ⁻ / Glu ⁻ host cell; b) culturing the transformed bacterial host cell under appropriate conditions; c) obtaining a host cell having a PTS ⁻ / Glu ⁺ phenotype by expression of a glucose assimilation protein; And d) obtaining an increased amount of product in the transformed bacterial host cell compared to the amount of product desired in PTS bacterial cells cultured under essentially the same culture conditions, wherein the desired product is pyruvate, PEP , Lactate, acetate, glycerol, succinate, ethanol and corismate. In a first embodiment, the host cell is selected from the group consisting of E. coli cells, Bacillus cells and Pantoea cells. In a second embodiment, the glucose assimilation protein is a galactose permease obtained from E. coli or a glucose transporter having at least 80% sequence homology with it. In a third embodiment, the glucose assimilation protein is a glucokinase obtained from E. coli or a glucokinase having at least 70% sequence homology with it.

In a fourth aspect, the invention features the original carbohydrate transport PTS available in the PTS for the ^- / Glu ^- includes that which steps of a method for increasing the carbon flow into the pathway of a host bacterial cell: a) Transforming PTS ⁻ / Glu ⁻ host cells using a promoter and a first DNA construct comprising a contiguous DNA sequence corresponding to the upstream (5 ′) region of galactose permease, thereby transforming the PTS ⁻ / Glu ⁻ host cells into the PTS ⁻ / Glu ⁻ host cells. Altering an endogenous chromosomal regulatory region operably linked to a nucleic acid encoding galactose permease; b) a promoter, and glucosidase using the 2 DNA construct comprising a contiguous DNA sequence homologous to the upstream (5 ') region of the kinase, PTS ^- / Glu ^- by transforming a host cell, PTS ^- / Glu ^- host cells Altering an endogenous chromosome regulatory region, operably linked to a nucleic acid encoding a glucokinase in it; c) integrating the first and second DNA constructs, wherein the first DNA construct replaces the endogenous promoter of the nucleic acid encoding galactose permease, and the second DNA construct replaces the endogenous promoter of the nucleic acid encoding the glucokinase. Where both galactose permease and glucokinase are expressed in the host cell, wherein said expression is compared to the metabolic pathway of the transformed host cell in comparison to the carbon flow to the same metabolic pathway in the corresponding unmodified PTS ⁻ / Glu ⁻ bacterial cells. Causing increased carbon inflow. In a first embodiment, the metabolic pathway is a conventional aromatic pathway. In a second embodiment, the method is more trans Kane Tortola azepin trans aldolase and phosphorylation to play pyruvate PTS using a polynucleotide encoding a protein selected from the group consisting of a synthase in ^- / Glu ^- host cells The transformation step of the.

Fifth aspect, the invention is Glu ⁺ phenotype to, be phosphoric transition using the enzyme transport system (PTS) PTS in order of the original carbohydrate transport ^- / Glu ^- relates to a process for recovering a bacterial host cell, which comprises the following A) transforming PTS ⁻ / Glu ⁻ host cells using a first DNA structure comprising a promoter and a contiguous DNA sequence corresponding to the upstream (5 ′) region of the glucose transporter, thereby converting PTS ⁻ / Glu ⁻ Altering an endogenous chromosomal regulatory region operably linked to a nucleic acid encoding a glucose transporter in a host cell; b) incorporating the first DNA construct, wherein the first DNA construct replaces the endogenous promoter of the nucleic acid encoding the glucose transporter; c) Expression of glucose transporter, wherein said expression restores the Glu ⁺ phenotype to PTS ⁻ / Glu ⁻ host cells. In a preferred embodiment, the method according to this aspect also uses a second DNA construct comprising an exogenous promoter and a contiguous DNA sequence corresponding to the upstream (5 ′) region of the glucokinase, PTS ⁻ / Glu ⁻ host cell the transformed, by switching PTS ^- / Glu ^- glucosidase variant of the host, which is connected to the endogenous chromosomal nucleic acid encoding the kinase operatively controlled within the cell area; Allowing integration of a second DNA structure that replaces the endogenous promoter of a nucleic acid encoding a glucokinase; And allowing expression of glucokinase. In one embodiment, the restored Glu ⁺ cells have a specific growth rate of at least about 0.4 hours ⁻¹ . In another embodiment, the glucose transporter is galactose permease.

In a sixth aspect, the present invention relates to a method of increasing the availability of phosphoenolpyruvate (PEP) in bacterial host cells, which comprises the following steps: a) phosphatase for native carbohydrate transport Selection of a bacterial host cell having a PTS ⁻ / Glu ⁻ phenotype, which may use a transport system (PTS); b) modifying an endogenous chromosomal regulatory sequence of a selected bacterial host cell comprising transformation of said selected bacterial host cell using a DNA construct comprising a promoter, wherein said DNA structure works with a nucleic acid encoding a glucose assimilation protein Chromosomally integrated into selected bacterial host cells that replace endogenously linked endogenous promoters; c) culturing the transformed bacterial host cell under appropriate conditions; And d) obtaining an unaltered host cell having a PTS ⁻ / Glu ⁺ phenotype with expression of glucose assimilation protein, wherein the PEP utilization is essentially equivalent to the PEP utilization in corresponding unaltered PTS bacterial host cells cultured under the same culture conditions. Increased compared to. In one embodiment, the glucose assimilation protein is a DNA construct comprising a galactose permease and an exogenous promoter replacing the endogenous promoter of galactose permease. In another embodiment, the glucose assimilation protein is a DNA construct comprising a glucokinase and an exogenous promoter replacing an endogenous promoter of glucokinase.

In an eighth aspect, the present invention relates to a method of increasing carbon flow into the metabolic pathway of PTS ⁻ / Glu ⁻ bacterial host cells that may originally use a phosphatase transport system (PTS) for carbohydrate transport, And a DNA construct comprising an exogenous promoter and a contiguous DNA sequence corresponding to the upstream (5 ′) region of galactose permease, thereby transforming the PTS ⁻ / Glu ⁻ host cell, thereby producing a PTS ⁻ / Glu ^- changing an endogenous chromosomal regulatory region operably linked to a nucleic acid encoding galactose permease in a host cell; b) an exogenous promoter (promoter), and the second using a DNA construct, PTS including the adjacent DNA sequence homologous to the upstream (5 ') region of glucokinase ^- / Glu ^- by transforming a host cell, PTS ^- / Glu ^- changing an endogenous chromosome regulatory region operably linked to a nucleic acid encoding a glucokinase in a host cell; c) integrating the first and second DNA constructs, wherein the first DNA construct replaces the endogenous promoter of the nucleic acid encoding galactose permease, and the second DNA construct replaces the endogenous promoter of the nucleic acid encoding the glucokinase. ; d) culturing the transformed bacterial host cells under appropriate conditions; And e) the expression of galactose permease and glucokinase from the modified regulatory region has an increased growth rate compared to the specific growth rate of the unaltered corresponding PTS bacterial host cells cultured under essentially the same culture conditions. Obtaining altered bacterial cells.

Includes that which steps of a method of increasing the production of desirable products in the E. coli host cell in terms of the ninth present invention is the original carbohydrate transport the PTS that can be used for the PTS ^{- -} / Glu: a ) using the promoter and, second, which contains a contiguous DNA sequence homologous to the upstream (5 ') region of the enzyme galactose transmission 1 DNA construct E. coli PTS ^- / Glu ^- by transforming the cells, E. coli PTS ^- / Glu ^- modifying an endogenous chromosome regulatory region operably linked to a nucleic acid encoding galactose permease in the cell; b) an exogenous promoter and, by using the DNA construct of claim 2, which includes a contiguous DNA sequence homologous to the upstream (5 ') region of the glucokinase E. coli PTS ^- / Glu ^- by transforming the cells, E. coli PTS ^- / Glu ^- the step of modifying the endogenous chromosomal regulatory regions that are operably coupled to the nucleic acid encoding the glucokinase in the cells; c) culturing E. coli PTS ⁻ / Glu ⁻ cells transformed under appropriate conditions to allow for expression of galactose permease and glucokinase; d) a corresponding PTS is cultured under the same culture conditions in essence ^- / Glu ^- E. coli by the wind compared to the amount of the probable products in the cells, and transformed E. coli of step increased yield the desired product in the cell, wherein the desired product Are ethanol, corismate and succinate.

In a tenth aspect, the invention relates to a transformed bacterial cell obtained according to the first to ninth aspects. In one preferred embodiment, the transformed bacterial cells are E. coli cells, Bacillus cells or Pantoea cells.

Brief description of the drawings

1A and 1B illustrate the pathway of central carbon metabolism in E. coli showing the induction of the carbon backbone of various preferred compounds, including those of amino acid biosynthesis processes. From FIG. 1A, it can be observed that (a) glucose is transported across cell membranes by galactose permease (GalP) transport protein and (b) glucose travels across membranes to be phosphorylated by PEP in the PTS system. have. Phosphorylation of glucose by PTS is a major consumer of PEP in PTS cells, and the percentages shown in the figure are: Holms (1986) The central metabolic pathways of Escherichia coli: relationship between flux and control at a branch point, efficiency of conversion to biomass, and excretion of acetate. In: Current Topics in Cellular Regulation, Vol. 28, pp. 69-105 Academic Press, New York.]. For example, 66% of the generated PEP is used in the PTS system. Subsequent metabolic systems are illustrated schematically in the figure: Embden-Meyerhoff pathway (appropriate process), pentose phosphate pathway, TCA pathway, conventional aromatic pathway, and Entner-Doudoroff pathway.

The following abbreviations have been used throughout the figures and specification: PEP = phosphoenolpyruvate; DAHP = 3-dioxy-D-arabino-heptulsonate 7-phosphate; DHQ = 3-dihydroquinate; DHS = 3-dihydroscimate; SHK = Sikmate; S3P = Sikmate 3-phosphate; EPSP = 5-enolpyrubil siemate 3-phosphate; PHE = phenylalanine; TYR = tyrosine; TRP = tryptophan; Pyk = pyruvate kinase, which is encoded by the pyk gene; And Ppc = PEP carboxylase, which is encoded by the ppc gene. In addition, the following genes are exemplified for conventional aromatic pathways: aro B encoding DHQ synthase; Aro D encoding DHQ dehydratase; Aro E, which encodes a sham dehydrogenase; Aro L and aro K, which encodes sham kinases; EPSP Although not specifically illustrated in aro C. encoding the aro A and koriseu mate synthase encoding the synthase, one of ordinary skill in the art erythritol Los -4P (E4P) and the aro G, aro F and H in the E. coli aro It is known that it encodes three isozymes of DAHP synthase that catalyze the conversion of PEP to DAHP. 1B illustrates various compounds, the preparation of which can be enhanced by increasing carbon flow and PEP utilization according to the method according to the invention.

2 illustrates the DNA sequence of a Galp-ptrc DNA cassette placed forward in SEQ ID NO: 1 cloned into an R6K vector (provides pR6K-galP). Italic and bold nucleotide sequences represent loxP sequences; The nucleotide sequences in bold and underline represent the chloramphenicol acetyltransferase (CAT) gene located reverse to gal P; Underline sequence represents the region -35 (TTGACA) and -10 region (TATAAT) of the trc promoter, the nucleotides in italics represents the operation of the character lac promoter trc; Bold nucleotides represent gal P ATG start codons (see Example 1B).

3 illustrates the DNA sequence (SEQ ID NO: 2) of the galP-trc DNA cassette after removal of the CAT gene. Bold nucleotides represent the gal P upstream sequence; Italic nucleotides represent single loxP sites, and bold and italic nucleotides represent trc promoter regions, where -35 boxes and -10 boxes are underlined (see Example 1E).

4 illustrates the DNA sequence of a glk-trc DNA cassette placed forward in SEQ ID NO: 3, which is usually cloned into an R6K vector. Italic and bold nucleotide sequences represent loxP sequences; The nucleotide sequences in bold and underline represent CAT genes in reverse arrangement in glk ; The underlined sequences represent the -35 region (CTGACA) and -10 region (TATAAT) of the trc induction promoter; Italic nucleotides represent the lac effector of the trc promoter; Bold nucleotides represent glk ATG start codons (see Example 1F).

5 illustrates a plasmid map of pMCGG, see Example 1G. The gal P and glk open reading frames (orf) are cloned into pACYC177, respectively, under the control of the trc promoter. Trc = trc promoter; gal P = galactose permease coding sequence; glk = glucokinase coding sequence; KmR '= (blocked by cloned gene) kanamycin resistance marker of pACYC177; Ampicillin resistance marker of Amp = pACYC177 and ori = plasmid origin of replication.

6 illustrates a plasmid map of pTrcm42, see Example 1A. LoxP = loxP site; trc = trc promoter, laclq = gene encoding Lacl ^q inhibitor protein; CAT = CAT coding gene; 5S = 5StRNA; rrnBT1 and 2 = ribosome RNA terminators; Amp = ampicillin resistance gene of pTrc99a; And ori = pMB1 origin of replication.

7A and 7B. 7A shows a diagram of a schematic promoter DNA integration cassette comprising loxP-CAT-loxP-ptrc. The DNA homologous portion is added to the site where integration is desired by PCR so as to be adjacent to the entire cassette, see Examples 1E and 1F. loxP = loxP site; CAT = chloramphenicol acetyltransferase gene; trc = trc promoter ; And ATG = initiation codon of the glucose assimilation gene targeted for integration of the DNA cassette. 7B illustrates a plasmid map of pR6K-galP-trc, which comprises 221-183 bp upstream of the ATG start codon of gal P (5 'portion) and ATG start codon of gal P (3' portion) and 40 bp Provided by amplifying the DNA cassette of FIG. 7A adjacent 40-bp homology to the regulatory region of gal P from upstream, wherein ploxP = loxP encoded plasmid; loxP = introduced loxP sequence from DNA cassette; trc = trc promoter; Km = kanamycin resistance gene; CAT is as defined above; ori = R6K plasmid origin of replication.

8 illustrates the nucleotide sequence of plasmid pSYCO101 (SEQ ID NO: 4).

9 shows a plasmid map of pSYCO101, where DAR1 (dihydroxyacetone phosphate reductase) and GPP2 (glycerol-phosphate phosphatase) are glycerol pathway genes; STR (R) is a spectinomycin resistance coding gene; pSC101 ori is the origin of replication of the plasmid; The term AspA is an aspartate ammonia lyase gene terminator; dhaB1-3, dhaX, and orf W, X, Y are 1,3-propanediol pathway genes; atten is an E. coli threonine operon attenuator; The term TonB is an E. coli tonB gene terminator; trc is the trc promoter and EcoR1 is the EcoR1 restriction enzyme site in pSYCO101 (see Example 1H).

10A and 10B show glycerol and 1,3-propanediol for cell growth and fermentation time (h) using plasmid encoded by PEP-independent glucose transport system in PTS ⁻ / Glu ⁺ E. coli . The preparation is illustrated. PTS ^- / Glu ^- strain, KLpts7 (Example 1D) is pMCGG (Example 1G), and is transformed by using the pSYCO101, a strain generated as a result is for cell growth, and glycerol production of 1, 3-propanediol Tested and analyzed. What is the optical density (OD ₆₀₀ )? ◆? It is represented by, and the glycerol concentration is? ▲? 1,3-propanediol concentration is represented by? ■? It is represented by

11A and 11B show fermentation time (h) in PTS ⁻ / Glu ⁺ E. coli using cell growth and expression of gal P regulated by chromosomally integrated trc promoter and expression of glk by natural regulation. The preparation of glycerol and 1,3-propanediol for is illustrated. PTS ⁻ / Glu ⁻ , KLpts7 (Example 1D), Glu ⁺ is made by incorporation of the trc promoter at the gal P target site to provide strain KlgalP-ptrc (Example 1E). The strain was transformed using pSYCO103 and analyzed by fermentation (Example 2). The parameters tested were the cell density (OD ₆₀₀ ) (? ◆?) In FIG. 11A and the glycerol concentration (? ▲?) And 1,3-propanediol concentration (? ■?) In FIG. 11B.

12A and 12B show glycerol and 1, for fermentation time (h) in PTS ⁻ / Glu ⁺ E. coli using cell growth and expression of gal P and glk regulated by chromosomalally integrated trc promoters. The preparation of 3-propanediol is illustrated. PTS ⁻ / Glu ⁻ , KLpts7 (Example 1D) were made Glu ⁺ by integration of the trc promoter at gal P and glk target sites to obtain strain KLGG (Example 1F). The strain was transformed with pSYCO101 and analyzed by fermentation (Example 3). The parameters tested are the cell density (OD ₆₀₀ ) (? ◆?) In FIG. 12A and the glycerol concentration (? ▲?) And 1,3-propanediol concentration (? ■?) In FIG. 12B.

13 is a map of plasmid pVHGalPglk11 as described in Example 1G.

14A-E illustrate the nucleotide sequence (SEQ ID NO: 25) and the coding sequence for Glk (SEQ ID NO: 26) of galP and pVHGalPglk11.

Detailed description of the preferred embodiment

Unless otherwise indicated, the performance of the present invention will be carried out by conventional techniques of molecular biology (including recombination techniques), microbiology, cell biology, and biochemistry in the art. Such techniques are explained fully in the literature, for example MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Laboratory Press; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al., 1987 and published annually); GENE EXPRESSION TECHNOLOGY, (Goeddel, D. et al., 1991, METHODS IN ENZYMOLOGY Vol. 185 Academic Press, San Diego, CA); GUIDE To PROTEIN PURIFICATION (Deutshcer M. P. et al., 1989, METHODS IN ENZYMOLOGY, Academic Press, San Diego, CA); PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (Innis et al., 1990, Academic Press, San Diego, CA); OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait et al., 1984); PCR: THE POLYMERASE CHAIN REACTION, (Mullis et al., 1994); MANUAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY, 2nd Edition (ed by A.L. Demain et al., 1999); MANUAL OF METHODS FOR GENERAL BACTERIOLOGY (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, et al.), Pp. 210-213 American Society for Microbiology, Washington, DC .; And BIOTECHNOLOGY: A TEXTBOOK OF INDUSTRIAL MICROBIOLOGY, (Thomas D. Brock) 2nd Edition (1989) Sinauer Associates, Inc., Sunderland, Mass.

Justice

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of this invention. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd edition, John Wiley and Sons, New York (1994) and Hale and Marham, THE HARPER DICTIONARY OF BIOLOGY, Harper Perennial, New York (1991) are used in the present invention. As a general dictionary of many terms.

The numerical range contains the numbers that define the range. Unless defined otherwise, nucleic acids describe the 5 'to 3' orientation from left to right, and the amino acid sequences describe the carboxy orientation from left to right in each amino group.

The introductory description provided herein is not intended to limit the various aspects or embodiments of the invention, which may be all mentioned in the specification. Accordingly, the terms defined immediately below are more fully defined by reference throughout the specification.

References, published patents, and pending patent applications cited herein are hereby incorporated by reference.

The phosphotransferase system (PTS) (EC 2.7.1.69) is referred to as the phosphoenolpyruvic acid-dependent sugar intake system. It is a system of transport proteins that participate in the transport of various sugars and in phosphoenolpyruvate dependent phosphorylation. The system contains two phosphoproteins, enzyme I and HPr, which are common to all PTS carbohydrates and catalyze the transport of the phosphoryl group of PEP to carbohydrate specific membranes to which enzyme II is bound and then to carbohydrates. In some cases, enzyme III is located between HPr and enzyme II. To distinguish between various enzymes II and III, the tridentate superscript is often used to indicate that carbohydrates are the preferred substrate. For example, enzyme II ^glc means that glucose is the preferred substrate. However, other substrates may be used.

The terms "Ptsl" and "enzyme I" mean the phosphotransportase EC 2.7.3.9 encoded by ptsl in E. coli . The terms "HPr" and "PtsH" refer to phosphocarrier proteins encoded by ptsH in E. coli . "Glucose-specific IIA member", "enzyme II ^glc " and "Crr" mean EC 2.7.1.69 encoded by crr in E. coli . The PTS includes Ptsl, PtsH and Crr and functionally equivalent proteins.

The term "PTS ^- / GIu ^_ phenotype" and "PTS ^- / Glu ^-" means a host cell is inactivated compared to wild-type cells that PEP- PTS PTS-dependent response, the ability to use glucose as a carbon source significantly reduced do. Effectively, less PEP is used for the transport of glucose than wild type PTS cells.

By "glucose ⁺ (Glu ⁺ ) phenotype recovery" is meant a host cell capable of using glucose as a carbon source despite the inactivation of PTS. Add used herein refers to a host cell ^- "PTS ^- / Glu ⁺ phenotype" is a PTS with the restored Glu ⁺ phenotype ^- / GIu.

"Increased phosphoenolpyruvic acid (PEP) availability" refers to an increase in the total amount of PEP in a cell whose carbon number increases with metabolic or productive pathways; otherwise, the PEP is a PTS for the phosphorylation of glucose. Is metabolized within.

The phrase "increased carbon flow" means an increase in the availability of carbon substrates in metabolic or productive pathways, in other words, the carbon substrates are converted by the metabolism of PEP in PTS. Carbon flow in individual routes can be measured by well known methods such as gas chromatography and mass spectrometry. The carbon flow measured in the product may be at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, or greater than the carbon flow in the corresponding PTS cells growing under essentially the same growth conditions.

"Specific growth rate (μ)" means an increase in mass or cell number per hour. In one embodiment of the invention, cells with a restored Glu ⁺ phenotype are at least about 0.3 hr ⁻¹ , at least 0.4 hr ⁻¹ , at least 0.5 hr ⁻¹ , at least 0.6 hr ⁻¹ , at least 0.7 hr ⁻ when grown on glucose. Will have a specific growth rate (μ) of ¹ and at least 0.8 hr ⁻¹ .

"Regulatory region" and "regulatory sequence" refer to a nucleic acid sequence that is used interchangeably herein and is operably linked to a coding region of a gene that affects expression of the coding region. Regulatory sequences may inhibit, inhibit or promote the translation of mRNA or the expression of operably linked coding sequences. Examples of regulatory sequences include promoters, enhancers, ribosomal binding sites, effectors, and silencers.

"Endogenous chromosome regulatory region" and "homologous chromosomal regulatory region" refer to chromosomal regulatory regions, which are naturally occurring in host cells and operably linked to polynucleotides encoding glucose assimilation proteins in the host cell.

As used herein, “promoter” refers to a regulatory nucleic acid sequence that functions in direct transcription of a downstream gene or genes. The promoter according to the present invention has two consensus regions. The first consensus region is located about 10 base pairs above the transcription initiation region and is referred to as -10 consensus region (also -10 box or Pribnow box). The second consensus region is located about 35 base pairs from the initiation region and is referred to as -35 consensus box or sequence. The linker or spacer sequence is located between matched boxes and generally comprises 14 to 20 bp.

As used herein, an “exogenous promoter” is a promoter that is different from a naturally occurring promoter and is operably linked to an endogenous coding region of glucose assimilation protein in a host cell, and comprises a non-native promoter, a synthetic promoter, Including but not limited to modified naturally occurring promoters. Modified naturally occurring promoters include naturally occurring endogenous promoters that are operably linked to polynucleotides encoding glucose assimilation proteins, wherein the native promoters are altered and subsequently reintroduced into host cell chromosomes and endogenous coding regions of glucose assimilation proteins. Natural endogenous promoters that are not operably linked to.

"Inducible promoter", "modified promoter" and "variant promoter" refer to a promoter, wherein one or more nucleotides are modified. In one preferred embodiment, the inducible promoter will comprise one or more nucleotides of the promoter -35 box making modifications such as substitution.

It is well understood by those skilled in the art that "transcription control conditions" or "transcriptionally regulated" indicates that the transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on operably linked to a member that contributes or promotes transcription initiation.

"Operationally linked" means a parallel state in an arrangement that allows members to be functionally connected to each other. For example, if it commands transcription of a sequence, the promoter is operably linked to the coding sequence.

"Under translation control" is a term well understood to those skilled in the art that refers to the regulatory process that occurs after messenger RNA is formed.

As used herein, the term "gene" refers to a DNA segment involved in polypeptide production and encompasses not only the coding region but also the region before and after the sequence (intron) interposed between each coding segment (exon).

As used herein, “polynucleotide” and “nucleic acid” refer to a polymeric form of nucleotides of any length, either ribonucleotide or deoxyribonucleotide. The term refers to single-, double- or triple-helix DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases, or other natural, chemical, biochemically modified, non- -Include natural or derived nucleotide bases. The following are non-limiting examples of polynucleotides: genes or gene fragments, exons, introns, mRNAs, tRNAs, rRNAs, ribozymes, cDNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence , Isolated RNA, nucleic acid probes, DNA constructs and primers of any sequence. Polynucleotides may include methylated nucleotides and nucleotide analogues, modified nucleotides such as uracil, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches.

"Structural sequence" is the putative polynucleotide sequence of DNA encoding product formation. The structural sequence can be encoded such that the amino acid sequence of the polypeptide chain with messenger RNA becomes its primary product. Structural sequences may also encode RNA formation with structural or regulatory functions.

As used herein, the term "vector" refers to a polynucleotide construct designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, transport vectors, plasmids, cassettes and the like. In a special case of DNA cassettes, DNA can be produced in vitro by PCR or other suitable technique.

The term "overexpression" refers to increased levels of replication products of the same gene in a host cell.

"Protein" and "polypeptide" are used interchangeably herein. Amino acid subunit codes defined as identified by the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used throughout this disclosure. It will also be appreciated that the polypeptide may be encoded by one or more nucleotide sequences due to the retraction of the genetic code.

As used herein when describing a protein and the gene encoding it, the term for the gene is not capitalized and is italicized, for example glk A. The term for a protein is generally not italic and the first letter is capitalized, for example GlkA.

"Glucose assimilation protein" or "enzyme involved in glucose assimilation" means an enzyme or protein that can utilize a host cell using glucose as a carbon source. Enzymes and proteins involved in glucose assimilation include those involved in transporting glucose across cell membranes and those involved in phosphorylation of glucose, which is called glucose-6-phosphate.

"Phosphorylase" means an enzyme that catalyzes the reaction of glucose into which glucose becomes glucose-6-phosphate.

Known enzymes catalyzing the reaction include hexokinase (ECNo .: 2.7.1.1) and glucokinase (ECNo.2.7.1.2). Glucokinase is encoded by glk in an E. coil .

"Transport protein" or "transporter" means a protein that catalyzes the transport of molecules across cell membranes. In a preferred embodiment, the transporter, also referred to as permease, is a glucose transporter. Glucose transporters catalyze the active transport of glucose across the cell membrane into the cytoplasm. Glucose transporters can also catalyze the transport of other sugars. One example of a glucose transporter is galactose-protein cotransporter (also known as galactose permease), GalP. GalP is encoded by gal P in E. coli (Henderson et al. (1990) Phil. Trans.R . Soc. , London 326: 391-410).

"Active transport" means transport of a compound into a cell accompanied by energy consumption. One example encompassed by the present invention is the use of membrane potential by glucose transporters.

As used herein, "selectable label" means a gene capable of expression in a host cell that facilitates the selection of a host containing the introduced nucleic acid or vector. Examples of such selectable labels include, but are not limited to, antimicrobial agents (eg, kanamycin, erythromycin, actiomycin, chloramphenicol, spectinomycin, and tetracycline). For example, the "CM ^Rn " and "CAT" designations mean the same gene, chloramphenicol resistance gene and also known chloramphenicol acetyl transportase gene.

"Flanking sequences" means any sequences upstream or downstream of the sequences discussed (eg, genes A, B, and C; genes B are close to A and C gene sequences). In some embodiments, contiguous sequences are present on only one side (3 'or 5') of the DNA fragment, but in preferred embodiments, each side of the sequence discussed is contiguous.

The term wild type refers to an intact or naturally occurring host cell or host cell sequence.

As used herein, the term “endogenous” refers to a nucleic acid in a host or a protein encoded by said naturally occurring nucleic acid. The term “exogenous” refers to nucleic acids or proteins from different host cell strains. Exogenous sequences can be non-host sequences and synthetically modified native sequences.

The term "homologous" refers to the same organism, strain or species. A "homologous sequence" is a sequence found in the same genetic origin or species. For example, if a host strain lacks a particular gene, the gene is considered homologous if the same gene is found in different strains of the same species. The term “heterologous” refers to different organisms, strains or species, and more particularly to nucleic acid or amino acid sequences that are not naturally occurring in the host cell.

"Transformation", "transduction" and "infection" refer to the incorporation or introduction of new polynucleotides into a cell. The introduced polynucleotide can be incorporated into chromosomal DNA or introduced as another chromosomal replication sequence.

As used herein, the term “isolated” or “purified” refers to an enzyme, nucleic acid, protein, peptide or co-factor that has been removed from one or more members with which it is naturally associated.

As used herein, "purpose product" means the desired compound to which the carbon substrate is biotransformed. Exemplary objective products include succinate, lysine, glycerol, methionine, threonine, isoleucine, pyruvic acid, ethanol, formate, acetate, DAHP, DHQ, DHS, SHK, S3P, EPSP, corimate, phenylalanine, tyrosine, tryptophan, And asocorbic acid intermediates.

As used herein, the term "carbon source" includes suitable carbon substrates commonly used by microorganisms, for example hexose 6 is glucose (G), glucose, lactose, sorbose, fructose, eye Dose, galactose and mannose of both D or L form, or mixtures of hexasaccharide, such as glucose and fructose, and / or hexasaccharide acid. Preferred carbon substrates include glucose and fructose.

When referring to a gene or protein, the terms "non-functional", "inactivated" and "inactivation" mean eliminating or greatly reducing the known normal function or activity of the gene or protein. Methods such as deletions, mutations, substitutions, or blocking, insertions in inactivated nucleic acid sequences that render a gene or protein non-functional.

A “host cell” is a cell capable of receiving a heterologous polynucleotide introduced. In one embodiment, the host cell is a Gram-negative or Gram-positive bacterium.

The term “bacteria” as used herein refers to any group of microscopic organisms that are prokaryotes that lack membrane-bound nuclei and organelles. All bacteria are surrounded by lipid membranes that regulate the flow of matter inside and outside the cell. The rigid cell wall is completely surrounded by bacteria and lies outside the membrane. There are a number of different types of bacteria, including, but not limited to, strains of Bacillus, Streptomyces, Pseudomonas, and intestinal bacterial populations.

As used herein, the "intestinal bacteria" family refers to bacterial strains that have a general characteristic of gram negative and can live in an anaerobic state. Inside the Enterobacteriaceae family El Winiah (Erwinia), Enterobacter bacteria (Enterobacter), glucono bakteo (Gluconobacter), keulrep when Ella (Klebsiella), Escherichia (Escherichia) in, acetonitrile bakteo (Acetobacter), Koh Rene bacteria (Coyrnebacteria) and Pantoea is included.

According to the present invention, "change the bacterial host" or "host strain bacteria" is PTS ^- is a genetically engineered bacteria cells with / Glu ⁺ phenotype.

It refers to cells ^- "non-changing bacterial host cell" is a bacterial host cell or to use the PTS PTS for the transport and phosphorylation of glucose ^- / Glu.

As used herein, "chromosome integration" is the process by which introduced polynucleotides are incorporated into a host cell chromosome. The process preferably takes place by homologous recombination. Homologous recombination is the exchange of DNA fragments between two DNA molecules, wherein the homologous sequence of the polynucleotide introduced above is aligned with the homologous region of the host cell chromosome and the sequence between the homologous regions of the chromosome is introduced by double exchange. Replaced with a sequence of polynucleotides.

"Target site" means a predetermined genomic location within a host cell chromosome in which integration of a DNA construct occurs.

As used herein, "modified" levels of protein or enzyme activity produced by a host cell are meant to regulate the level of protein or enzyme activity produced during the cultivation, which level is increased or decreased as desired.

As used herein, when referring to a nucleic acid or polynucleotide, the term “modification” means that the nucleic acid has been altered in some way compared to the wild type nucleic acid, eg, mutation, substitution, insertion of all or part of a nucleic acid. , Deletion; Or operative linkage of the transcriptional regulatory region. As used herein, the term “mutation” when referring to a nucleic acid means any alteration in the nucleic acid that causes the product of the nucleic acid to be partially or wholly inactivated. Examples of mutations include, but are not limited to, point mutations, lattice shift mutations and partial or complete deletions of genes.

A polynucleotide or polypeptide having a certain proportion of "sequence homology" to another sequence (eg, 80%, 85%, 90%, 95%, 96%, 97% or 99%), when aligned, It means that the percentage of base or amino acid is the same by comparing the two sequences. The alignment and percentage homology or sequence homology is well known to those skilled in the art, for example CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al., 1987) Supplement 30, section 7.7.18. Decisions can be made using the software described in. Preferred programs are the GCG Pileup program, FASTA (Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85: 2444-2448) and BLAST (BLAST Manual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl Library Med (NCBI NLM), NIH, Bethesda MD and Altschul et al., (1997) NAR 25: 3389-3402. Another preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania), which preferably uses the following default parameters: mismatch = 2; open gap = 0; extend gap = 2. Another sequence software program that can be used is the TFastsA Data Searching Program, which can be used in Sequence Analysis Software Package Version 6.0 (Genetic Computer Group, University of Wisconsin, Madison, Wis.). Those skilled in the art will recognize that the sequences included in the present invention are also defined by the ability to hybridize under stringent conditions with the illustrated sequence.

If the single-stranded form of nucleic acid can anneal with other nucleic acids under appropriate conditions of temperature and solution ionic strength conditions, it can be "hybridized". Hybridization and washing conditions are known and illustrated in Sambrook 1989, supra (see chapters 9 and 11, respectively). Low stringency hybridization conditions correspond to a Tm of 55 ° C. (eg 5X SSC, 0.1% SDS, 0.25 milk and form-free or 5X SSC, 0.5% SDS and 30% formamide). do. Suitable stringent conditions correspond to 0.05% milk in the presence or absence of 6X SSC, 0.1% SDS, formamide, and stringent conditions correspond to, for example, Tm at 65 ° C and 0.1X SSC, 0.1% SDS.

It is well understood in the art that acidic derivatives of other mixtures, such as sugars and organic acids, may be present under various ionization conditions depending on their surrounding medium, whether in solution or outside of the solution in which they are prepared in solid form. For example, the use of terms such as gluconic acid or acetic acid assigned to these molecules is intended to cover all ionization states of the organic molecules mentioned. That is, for example, "gluconic acid" and "gluconate" refer to the same organic moiety and are not intended to specify a particular ionization state or chemical form.

As used herein, the term "culture" means fermentative bioconversion of the carbon substrate into the desired product in the reaction vessel. Bioconversion means contacting microorganisms with a carbon substrate to convert the carbon substrate to the desired product.

As used herein, the term “containing” and its etymology are used in their broad sense; It is a term having the same meaning as "comprising" and the same word as that.

"A", "an" and "the" include plural references unless the context clearly dictates otherwise.

", And so that the desired product produced from the carbon source in the production of the desired product is enhanced compared to the production of the desired product in the corresponding non-changing bacterial host cell" means that the PTS for the production of the desired product ^- / Glu ⁺ Contacting a bacterial cell with a substrate, such as a carbon source.

Preferred Embodiment

The present invention relates to a method of increasing carbon flow into a desired metabolic pathway of a host cell that can originally use PTS for carbohydrate transport, the method comprising: selecting a PTS ^- phosphorus host cell that is phenotypically effective. And modifying one or more homochromosomal regulatory regions operably linked to chromosomal nucleic acids encoding polypeptides involved in glucose assimilation to restore the glucose ⁺ phenotype, thereby increasing carbon flow into and through the desired metabolic pathways. It comprises the step of.

A. PTS Host Cells

A general overview of the PTS is described in Postma et al., 1993, Microbiol. Rev. 57: 543-594; Romano et al., 1979, J. Bacteriol . 139: 93-97 and Saier et al. 1990, In: BACTERIAL ENERGETICS pp. 273-299, TA Krulwich, Ed. Academic Press, NY]. Host cells or strains useful in the present invention include any individual who can utilize a PTS system for carbohydrate transport. This includes prokaryote belonging to the genus Escherichia , Corynebacterium , Brevibacterium , Bacillus , Pseudomonas , Streptomyces , Pantoea or Staphylococcus . A list of suitable individuals is shown in Table 1. If inactivation of the PTS occurred in any of these individuals, the carbon flow and PEP (and PEP precursors) in the cells would potentially increase for alternative metabolic pathways, resulting in desired compounds from the cells (eg , Aromatics) could increase production.

Preferred host strains are those known to be useful for producing aromatic compounds, including strains selected from the genus Escherichia , Corynebacterium , Brevibacterium , Pantoea and Bacillus . Pantoea genus includes, but is not limited to, all members known to those skilled in the art, including, but not limited to, P. citrea , P. ananatis , P. stewartii , P. agglomerans , P. punctata, and P. terrea . Useful Bacillus strains include B. subtilis , B. licheniformis , B. lentus , B. brevis , B. stearothermophilus , B. alkalophilus , B. amyloliquefaciens , B. coaglulans , B. ciculans , B. lautus and B. thuringiensis . .

B. PTS ^_ / Glu ^_  Host Cell Screening

PTS ^- cell selection can be performed using techniques available to those skilled in the art. Inactivation is described by PEP phosphorylation, Gachelin, G. (1969). Biochem. Biophys. Acta . 34: 382-387; Romero, et al., (1979) J. Bact. 139: 93-97; Or Bouvet and Grimont., (1987) Ann. Inst. PEP-dependent phosphorylation of 2-deoxy-D-glucose using the protocol described in Pasteur / Microbiol 138: 3-13 will effectively reduce to undetectable levels when measured. PEP phosphorylation assays are also useful for determining PTS ^- expression levels.

PTS ⁻ / Glu ⁻ host cells can be selected from PTS wild type host cells by inactivating one or more genes encoding all or part of the enzymes including the PTS. For example, in one embodiment, the PTS is inactivated by deleting one or more genes selected from the group consisting of pts I, pts H and crr , respectively, encoding the EI, HPr and IIA ^Glc proteins of the PTS (Postma , et al (1993) Microbiol. Rev. 57: 543-594). In other embodiments, two or more of the genes are inactivated. Nucleotide sequences of pts I, pts H and crr were determined (Saffen et al., (1987) J. Biol Chem . 262: 16241-16253; Fox et al., (1984) Biochem. Soc. Trans . 12: 155 Weigel et al., (1982) J. Biol. Chem . 257: 14461-14469 and DeReuse et al., (1988) J. Bacteriol 176: 3827-3837). In other embodiments, inactivation of all three genes pts I, pts H and crr into deletions will effectively reduce PEP phosphorylation to undetectable levels.

In general, the methodology used in the present invention to inactivate the PTS is as follows. It is known that in E. coli pts I, pts H and crr are linked together in one operon. (1984) by E. coli strain JM101 (Yanisch-Perron et al., Using generalized transduction methods described in EXPERIMENTS WITH GENE FUSIONS, pp 110-112, Cold Spring Harbor Laboratory Press, NY). (1985) Gene 33: 103-119) and ptsHIcrr operon in strain PB103 (Mascarenhas (1987) PCT WO / 87/01130) were inactivated. Transduction was performed using P1vir phage and the TP2811 strain (Levy et al., (1990) Gene 86: 27-33) was used as a donor of the ptsHIcrr deletion. The method was carried out in two steps. First, cell-free phage suspensions were prepared by propagating bacteriophages on TP2811 strain. In the TP2811 strain, most of the ptsHIcrr operon was deleted and kanamycin-resistance markers were inserted in the same DNA region (Levy et al., (1990) Gene 86: 27-33). The obtained P1vir lysate was able to transduce ptsHIcrr deletion and kanamycin resistance markers simultaneously. Second, these phages were used to infect recipient strains JM101 or PB103 and to plate transfectants by plating on MacConkey-glucose plates containing kanamycin. The plates were incubated at 37 ° C. for 16 hours, resulting in several white colonies.

The recipient strains (JM101 and PB103) are kanamycin sensitive and form red colonies on MacConkey-glucose plates. The MacConkey-glucose plate contains an indicator dye that can vary from white to crimson depending on pH. If cells can transport glucose at high speed, normally they will secrete organic acids to produce red colonies. If glucose transport is reduced or absent, cells will not produce organic acids and colonies will become white. This makes it possible to determine whether the host cell is glucose ⁺ phenotype or glucose ^- phenotype.

Transduction of the resulting kanamycin resistance colonies was white, indicating that the ability of these cells to assimilate glucose was affected, presumably due to the migration of the ptsHIcrr operon deletion. To confirm this hypothesis, transductors can be selected and seeded in minimal medium containing glucose as the only carbon source. After incubation (for 12 hours at 37 ° C.), the transductors would have been predicted that there would be no detectable cell growth and the PTS parent strains JM101 and PB103 would grow very well, see WO 96/34961. do. The results were observed, on the basis of this result, PTS of the JM101 ^- PTS of the title derivative as PB11, PB103 and ^- derivatives was indicated as NF6.

Another test for the absence of the PTS system is based on the fact that PTS ^- strains become resistant to the antibiotic fosfomycin (Cordaro et al., (1976) J. Bacteriol 128: 785-793). ).

The methodology is inactivated PTS (PTS ^- / Glu ^-), but the preferred means for providing, other methods may also be used. One additional non-limiting method includes inserting an inhibitor binding region operably linked to a gene encoding an expressed protein or modifying the expression of the gene to occur in the presence of a specific molecule. For example, the lac effector is a DNA sequence recognized by a Lac inhibitor. When the effector is located in an appropriate position relative to the promoter, if the inhibitor binds to the effector, it will interfere with the binding of RNA polymerase and thus disrupt gene transcription. The interference can be eliminated by adding the inducer IPTG (isopropyl-β-D-thiogalactosid). The level of inhibition and / or induction will depend on the strength of the promoter, the location and sequence of the effector, and the amount of the inhibitor and inducer (Muller J. et al., (1996) J. MoL Biol 257: 21 -29). The lac effector is used to modulate a number of promoters, among them several variants of the lac promoter and the hybrid trc promoter.

Another non-limiting method of influencing the PTS ⁻ / Glu ⁻ phenotype involves inserting a promoter that affects the expression of the structural gene sequence when certain conditions are present. For example, Pm promoters from TOL plasmids can be used to regulate gene expression. In this system, gene expression occurs when benzoate or toluate is added to the medium. (Mermod et al., (1986) J. Bact . 167: 447-454). Another additional non-limiting method of influencing the PTS ^- phenotype is Carrier and Keasling (1997) BiotechnoL Prog . 13: 699-708 to alter mRNA stability.

However, in order to increase carbon flow or redirect the carbon flow to the desired metabolic pathway in inactivated PTS host cells, one must amplify or stop regulating glucose transport and phosphorylation.

C. Glucose ⁺  Phenotype recovery

While not wishing to be bound by theory, the modification of alternative glucose assimilation pathways offsets the inability to actively transport glucose by PTS, resulting in PEPs metabolizing host cells differently in glucose transport for other purposes. It becomes available.

Once a PTS ⁻ / Glu ⁻ host cell is obtained, the homologous chromosome regulatory region operably linked to the chromosomal nucleic acid encoding the polypeptides involved in glucose assimilation is modified to restore the glucose ⁺ phenotype, thereby restoring the PTS ⁻ / Glu ⁺ phenotype. To obtain. The regulatory region operably linked to the expression of the polypeptide involved in glucose assimilation may be an effector, a promoter, an inhibitor or an inhibitor.

In one preferred embodiment, a DNA cassette containing the control region comprising a promoter PTS ^- the introduction into the host cell, and connected to the chromosomal nucleic acid encoding a polypeptide involved in the glucose assimilation operatively homologous chromosomes regulatory region ^- / Glu Modify to restore the glucose ⁺ phenotype.

D. Construct DNA Integration Cassettes Containing Regulatory Regions

, PTS according to the present invention typically ^- / Glu ^- a useful DNA constructs or cassettes endogenous chromosomal regulatory regions in the host strain, there may be mentioned a control sequence of the promoter. In another embodiment, the DNA cassette further comprises a selection marker and a sequence that enables autonomous replication or chromosomal integration, such as a recombinase recognition site. In another embodiment, the DNA cassette further comprises flanking sequences located upstream (5 ') and downstream (3') of the recombinase recognition site.

Promoter

In one embodiment, the regulatory region comprising the DNA cassette comprises a promoter. In further embodiments, the promoter is an exogenous promoter. In other embodiments, the exogenous promoter is a non-natural promoter and derivatives thereof. In further embodiments, the promoter is a natural promoter that is not operably linked to the polynucleotide encoding the glucose assimilation protein in its native endogenous form. In some embodiments, the exogenous promoter is a modified naturally occurring promoter whose original endogenous form is linked to a polynucleotide encoding a glucose assimilation protein, wherein the native promoter is altered and then the host cell chromosome. Reintroduced into For example, the original promoter may have been modified in the -35 region, the -10 region or the linker region, or the original promoter may comprise modification of the inhibitor binding site. In other embodiments, the original promoter is one that is not linked to a polynucleotide, more specifically galP , encoding a glucose transporter, such as a galactose-proton cocarrier . Further, in other embodiments, the original promoter is not linked to a polynucleotide encoding a phosphorylated protein such as glucokinase, more specifically to glk . Regulatory regions, including regulatory regions and particularly promoters useful in the DNA cassettes according to the invention, comprise sequences of about 20 to 200 bp, about 20 to 150 bp and about 20 to 100 bp.

Preferably, the promoter will be more potent than the naturally occurring endogenous wild type promoter and will result in increased expression of the glucose assimilation protein. Those skilled in the art will appreciate that various methodologies are known for determining the relative strength of such promoters. Promoter strength can be quantified using an in vitro method that measures the rate at which RNA polymerase binds to a particular DNA fragment, which also allows the measurement of transcription initiation (Hawley DK et al., Chapter 3: in PROMOTERS: STRUCTURE AND FUNCTION.RL / Rodriguez and MJ Chamberlin, Praeger Scientific.New York. In vivo methods may also be used to quantify promoter strength. For example, after the promoter is fused to the reporter gene, the efficiency of the RNA synthase can be measured. Deuschle et al. (1986) ( EMBO J. 5: 2987-2994) measured the intensity of 14 E. coli promoters using three different reporter genes. These promoters include: trc, tacI, D / E20, H207, N25, G25, J5, A1, A2, A3, L, Lac, LacUV5, Con, β-lactamase (bla), T5 "initial" P _L , and H / McC. Each of these promoters or derivatives thereof can be used as exogenous promoters in accordance with the present invention.

In addition, in its native endogenous form, modified naturally occurring promoters and native promoters which are not linked to polynucleotides encoding glucose assimilation proteins can be used according to the invention. The various promoters can determine the original promoters. Without wishing to be limited to this, in one embodiment, computer-based search algorithms can be used to identify sequences of specific genomes and to identify sequences presumed to be promoters. For example, one genomic region can be sequenced and analyzed for the presence of putative promoters using Neural Network for Promoter Prediction software (NNPP). The NNPP is a time delay network consisting mainly of two characteristic layers: a layer that recognizes a TATA box and a layer that recognizes a so-called "initiator", which is an area that extends the transcription start site. All of these characteristic layers are combined in one production device. The putative sequences can then be cloned into a cassette suitable for preliminary characterization and / or direct characterization in E. coli . In another embodiment, identification of consensus promoter sequences can be identified using homology analysis, eg, BLAST. The putative promoter sequence can then be cloned into a cassette suitable for preliminary characterization in E. coli .

Although many promoters and derivatives thereof can be used, preferred promoters include trc promoters and derivatives thereof (Amann et al., (1983) Gene 25: 167-178). The trc promoter is shown in Figure 2, where -35 box is TTGACA and -10 box is TATAAT. Another preferred promoter is the tac promoter. The nucleic acid sequences of the tac promoter and trc promoter are identical except for the linker region. The linker region of the tac promoter differs by 1 bp (Russell and Bennett (1982) Gene 20: 231-243). Another preferred promoter is the glucose isomerase (GI) promoter (also known as the xylose isomerase promoter). Amore et al. (1989) Appl. Microbiol. Biotechnol. 30: 351-357. The sequence of the fragment of the GI promoter (+50 to -7 in -10 boxes) is SEQ ID NO: 5

Where -35 boxes are represented by TTGACA and -10 boxes are represented by AATAAT.

Derivative promoters can include modifications to one or more nucleotides in positions corresponding to one nucleotide in a -35 box, linker region, or -10 box. In a preferred embodiment, the positions corresponding to positions in the -35 box in these derivative promoters are altered. Particularly preferred derivative promoters include modifications to the -35 boxes corresponding to TTGACA and TTTACA. Some TTGACA variants include TTGAAA, TTCAC and CTGACA. One special variant is for the position corresponding to the -35 position. Particularly preferred derivative promoters also include modifications to the -10 boxes corresponding to TATAAT, TAAGAT and TATGTT. Linker regions may also be modified (Burr et al., (2000) NAR 28: 1864-1870). Preferably, the linker region is 14 to 20 bp in length, preferably 16 bp, 17 bp, 18 bp and 19 bp, whether modified or not. Those skilled in the art are familiar with the methods used to effect modifications in the promoters and the present invention is not limited to these methods. One exemplary method is to use the Quikchange Kit (Stratagene); See also WO 98/07846; Russell and Bennett (1982) Gene 231-243 and Sommer et al. (2000) Microbiol . 146: 2643-2653.

Further preferred derivative promoters are trc derivative promoters. The trc derivative promoter may comprise one or more modifications within the -35 consensus box, -10 consensus box, or linker region. The modification may be insertion, substitution or deletion. In a preferred embodiment, the trc derivative promoter comprises one or more modifications to the -35 box. For example, in E. coli , since the codon at the -30 position is adenine (A), it can be substituted with thymine (T), guanine (G) and cytosine (C); The codon at position -31 is C, so that it can be substituted with A, T and G; The codon at position -32 is A, so it can be substituted with T, G and C; The codon at position -33 is G, so that it can be substituted by C, T and A; The codon at position -34 is T, so that it can be substituted by A, C and G; The codon at position -35 is T, so it can be substituted with A, G and C. One particularly preferred trc derivative promoter comprises a modification to the codon at position -35, most preferably where T is substituted with C.

Other preferred trc derivative promoters include modifications within -10 boxes. For example, since the nucleotide at -7 is T, it can be substituted with one nucleotide selected from the group consisting of C, G and A; Since the nucleotide at -8 is A, it can be substituted with 1 nucleotide selected from the group consisting of C, G and T; Since the nucleotide at -9 is G, it can be substituted with 1 nucleotide selected from the group consisting of C, T and A; Since the nucleotide at -10 is A, it can be substituted with 1 nucleotide selected from the group consisting of C, G and T; Since the nucleotide at -11 is A, it can be substituted with 1 nucleotide selected from the group consisting of T, G and C; Since the nucleotide at -12 is T, it can be substituted with 1 nucleotide selected from the group consisting of A, C and G.

Screening Markers and Recombinase Recognition Sites

The DNA cassette included in the present invention will include a selection marker, and a plurality of genes can be used to detect the gene insertion in E. coli . Some of these genes provide a selective phenotype. In this case, media conditions can be established such that only the colonies in which only the expression of these genes are activated proliferate. Other genes provide phenotypes that can be screened. Screenable phenotypes can often yield information regarding gene expression levels. Any desired marker can be used, but based on these properties, useful antibiotic resistance (Anb ^R ) markers include, but are not limited to, Cm ^R , Km ^R and Gm ^R. A preferred non-limiting example of a selection marker is the chloramphenicol acetyltransferase (CAT) gene.

In a preferred embodiment, the DNA cassette comprising the promoter to be inserted into the target site of the host cell chromosome will comprise selectable markers adjacent to either side of the recombinase recognition site. Recombinase sites are well known in the art and are generally divided into two distinct groups based on their catalytic mechanism, see Huang et al., (1991) NAR . 19: 443; Datsenko and Warner (2000) Proc. Natl. Acad. Sci 97: 6640-6645 and Nunes-Duby, D, et al, (1998) NAR 26: 391-406. Preferably, the recognition sites are identical.

One well known recombination system is the Saccharomyces Flp / FRT recombination system, which contains a Flp enzyme and two asymmetric 34 bp FRT minimal recombination sites (Zhu et al., (1995) J. Biol. Chem 270: 11646-) . 11653). The FRT site contains two 13 bp sequences that are inversion and incompletely repeated, which surrounds the 8 bp core asymmetric sequence where the crossing occurs (Huffman et al., (1999) J. Mol. Biol 286: 1-13).

One preferred recombinase system is a Cre / loxP site-specific recombination system of bacteriophage P1, which contains a Cre enzyme and two asymmetric 34 bp loxP recombination sites (Sternberg and Hamilton (1981) J. Mol. Biol . 150: 467-486); Palmeros, B, et al (2000) Gene 247: 255-264; Hoess et al. (1986) NAR 14: 2287-2300; Sauer B. (1994) Curr. Opinions in Biotechnol . 5: 521-527). The loxP region contains two 13 bp sequences that are inverted and incompletely repeated, which surrounds the 8 bp core asymmetric sequence where the crossing occurs. As a result of Cre-dependent intramolecular recombination between two parallel loxP sites, any intervening DNA sequences as circular molecules are cleaved, producing two recombinant products, each containing one loxP site (Kilby et al. , (1993) Trends Genet . 9: 414-421.

Homologous lateral region

The integrated DNA cassette according to the present invention may also comprise nucleic acid sequences homologous to the upstream (5 ′) region of the gene encoding the glucose assimilation protein. These homologous sequences preferably make up the first recombinase recognition site (5 'to it) and the promoter (3' to it) side. The nucleic acid sequence homologous to the upstream (5 ') region of the gene encoding the glucose assimilation protein is a) a 5' sequence for the endogenous regulatory region targeted for modification, and b) a 3 'of the endogenous regulatory region targeted for modification. Sequences derived from sequences. The 3 ′ sequence may comprise a portion of a glucose assimilation protein coding sequence. Homologous flanking sequences may comprise about 2 to 300 bp, about 5 to 200 bp, about 5 to 150 bp, about 5 to 100 bp and about 5 to 50 bp.

Gene and Glucose Assimilation Protein Isolation

Methods of obtaining the desired genes from bacterial cells are common and well known in the molecular biology art. For example, if the gene sequence is known, appropriate genomic libraries can be generated and screened. Once the sequences have been separated, various amounts of DNA can be obtained by restriction by amplifying the DNA using standard methods such as polymerase chain reaction (PCR) (USP 4,683,202). Sambrook et al., Cited above. See also. For the purposes of the present invention, upstream sequences as defined above of any gene encoding a glucose assimilation protein are suitable for use in the methods disclosed above.

In one embodiment, the gene encoding glucose anabolic protein is a glucose transporter. Transporters are disclosed in Saier et al., (1998) ADVANCES IN MICROBIAL PHYSIOLOGY, Poole, R. K., pp 81-136 Academic press, San Diego, CA. Typically, the glucose transporters are included in the Transport Council (TC) classification of class 2 transporters (GALP) and / or class 4 transporters (PTS) as defined herein.

One preferred glucose transporter is GalP, which is encoded by galP in E. coli . Those skilled in the art will recognize that genes encoding GalP isolated from a source other than E. coli would also be suitable for use in the present invention. In addition, it functions as a glucose transporter and 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% in GalP from E. coli Proteins having amino acid sequence homology of at least 85%, 90%, 95%, 97% and 98% will be suitable for use in accordance with the present invention.

Popularly available computer programs can also be used to determine sequences homologous to glucose anabolic proteins and in particular glucose transporters. Preferred programs include the GCG Pileup program, FASTA (Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85: 2444-2448) and BLAST (BLAST Manual, Altschul et al., Natl. Cent.Biotechnol.Inf. , Natl Library Med. (NCBI NLM), NIH, Bethesda MD and Altschul et al., (1997) NAR 25: 3389-3402).

Using BLAST, three or more permeases with similar protein sequences to GalP were found. For example, ara E with 64% protein sequence homology; Xyl E with 34% protein sequence homology and yaa U with 23% protein sequence homology. The permeases can also function as glucose transporters.

In many cases, the transporter proteins are highly regulated in the cell and in general the expression of the transporter genes is induced by the presence of a substrate in the medium. The glucose transporter, ie galactose permease (GalP) from E. coli , is induced by galactose, but a more preferred substrate is glucose (Henderson & Maidenn (1990). Phil. Trans. R. Soc. Lond. 326 : 391-410). In E. coli , in addition to GalP, other permeases can recognize and transport glucose, for example:

(a) High affinity galactose transport system encoded by the mglBCA gene (Hogg et al. (1991) Mol. Gen. Genet. 229: 453-459 and Ferenci T. (1996) FEMS Microbiol. Rev. 18: 301-317 ); And

(b) Mannose PTS systems, in which the membrane component PtsM has a broad substrate specificity and can transport glucose and fructose (Postma & Lengeler (1985) Microbial Rev. 49: 232-269 and Erni et al., (1987) J Biol.Chem . 262: 5238-5247).

In addition, the product of the ptsG gene, which is normally part of the glucose-PTS system, can be converted to PTC-independent transporters, which, by mutation, function as glucose assimilation proteins and more specifically as glucose-promoters. Ruijter et al (1992) J. Bact. 174: 2843-2850 and Erni et al (1986) J. Biol. Chem 261: 16398-16403.

In addition to the above well-characterized examples, other glucose transporters include those listed in the Transporter DB database. This is a relational database describing the predicted cytoplasmic transporter protein complement for organisms in which a complete genomic sequence is available (http://66.93.129.133/transporter/wb/index.html).

In another embodiment, the glucose assimilation protein is phosphorylated protein. The phosphorylated protein may be hexokinase, preferably glucokinase. One preferred glucokinase is Glk and see NCBI (NC 000913). As indicated above for glucose transporters, other glucose kinase enzymes can be identified using the computer programs such as FASTA, GCG Pileup and BLASTA.

E. coli is described in Flores et al. (2002) Met. Eng. 4: 124-137 and Curtis et al. (1975) J. Bacteriol. 122: 1189-1199 and other glucose kinase enzymes as suggested by the results. When glk was interrupted in E. coli , the cells had 22 to 32% residual glucose phosphorylation activity compared to wild type strains. BLAST searches of the E. coli genome using the Glk sequence did not show any protein with levels above 34% sequence homology. This may indicate that the measured glucokinase activity is governed by one or more enzyme (s), independent of or remote from Glk. Some of these glucose assimilation proteins that may contribute to glucokinase activity are listed below:

The list is not exhaustive and it has been suggested by the inventors that by using appropriate mutation-selection protocols, these and / or other kinases can be altered to increase their activity of phosphorylating glucose.

E. PTS ^-  / Glu ^-  Introduction of DNA Cassettes into Cells

Can be used to transform a host cell ^- Once the appropriate DNA cassette was constructed, they may be directly introduced into a suitable plasmid PTS ^- / Glu. Plasmids that can be used as vectors in bacterial organisms are well known and described in Manatis, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d Edition (1989) and MOLECULAR CLONING: A LABORATORY MANUAL, second edition (Sambrook et al. , 1989) and Bron, S, Chapter 3, Plasmids, in MOLECULAR BIOLOGY METHODS FOR BACILLUS, Ed. Harwood and Cutting, (1990) John Wiley & Sons Ltd.

Vectors useful in the present invention include vectors pSYCO101 (FIGS. 8 and 9), and pSYCO 101 derived plasmids pSYCO103 and pSYCO106; pKD46; pR6K-ECHO (Invitrogen); pJW168 (Palmeros et al., 2000 Gene , 247: 255-264); p trc M2; p trc 99A; pTrc99 (Pharmacia); pACYC177; pMCGG; pSC101; pKD46 (Datsenko and Wanner. (2000) PNAS 97: 6640-6645); And pKP32 (WO 01/012833).

Introduction of the DNA cassette and other vectors into the host cell can be accomplished by known transport techniques. Such gene transport techniques include transformation, transduction, conjugation and protoplast fusion. Gene transport is the process of transporting a gene or polynucleotide to one cell or cells where exogenously added DNA is taken up by bacteria. The general transformation process is taught in [CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Vol. 1, Ausubel et al., Edited by John Wiley & Sons, Inc. 1987, Chapter 9)] and using calcium phosphorylation methods, DEAE-dextran. Transformation and electroporation. Various transformation procedures are known by those skilled in the art to introduce nucleic acids into a given host cell. (USP 5,032,514; Potter H. (1988) Anal. Biochem 174: 361-373; Sambrook, J. et al. , MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press (1989); and Ferrari et al., Genetics, pgs 57-72 in BACILLUS, Harwood et al., Eds. Plenum Publishing Corp.).

PTS of the DNA cassette containing the promoter and the upstream sequence of the gene encoding the glucose assimilation protein ^- / Glu ^- introducing into the host cell, preferably resulting in a change in the endogenous chromosomal regulatory regions by replacement of the endogenous regulatory region . In one embodiment, the DNA cassette comprises an exogenous promoter and the promoter of the endogenous regulatory region is substituted. The introduced regulatory sequence (including promoter) is chromosomally integrated into PTS ⁻ / Glu ⁻ cells and the introduced sequence is operably linked to the coding sequence of the glucose assimilation protein replacing the endogenous regulatory sequence. In a preferred embodiment, the selectable marker gene, introduced with the integrated DNA cassette, is preferably [Palmeros et al. (2000) Gene 247: 255-264). Expression of the glucose assimilation protein, linked to the exogenous promoter, results in cells with a glucose ⁺ phenotype. Bacterial strains with a glucose ⁺ phenotype (PTS ⁻ / Glu ⁺ ) are also included in the present invention as disclosed herein.

In further embodiments, the altered endogenous regulatory region is a regulatory region of glucose transporters. In a preferred embodiment, the glucose transporter gene E. 60% to GalP from GalP and E. coli from the coli, 70%, 75%, 80%, 85%, 90%, 95%, 97% and 98 It encodes a glucose transporter having at least% amino acid sequence homology. In another embodiment, the altered endogenous regulatory region is a regulatory region of phosphorylated protein. In a preferred embodiment, the phosphorylated proteins glucokinase, more preferably 60% to Glk Glk from E. coli and from E. coli, 70%, 75% , 80%, 85%, 90%, 95 Phosphorylated proteins with amino acid sequence homology of at least%, 97% and 98%.

In one embodiment, said modified endogenous regulatory sequence operably linked to a glucose assimilation protein, in particular the modified endogenous regulatory sequence operably linked to galactose permease and the altered endogenous regulatory sequence operably linked to glucokinase according to the present invention. In the obtained PTS ⁻ / Glu ⁺ cells, production of the desired compound may be increased in comparison with wild type host cells. The desired compound includes the compounds illustrated in FIG. 1B. Particularly preferred compounds are dihydroxyacetone-P, glycerol, 1, 3-propanediol, pyruvate, lactate, chorismate, tryptophan, phenylalanine, tyrosine, oxalacetic acid, aspartic acid, asparagine, tyrosine, succinic acid, ethanol and acetyl Contains CoA. Particularly desired compounds include pyruvic acid, corismate and succinic acid.

F. PTS ^- / Glu ⁺  Further alteration of cells

PTS ⁻ / Glu ⁺ cells obtained according to the methods of the invention may further comprise heterologous polynucleotides encoding one or more proteins that direct carbon flow into and through the general directional pathway. The heterologous polynucleotide may be introduced into PTS ⁻ / Glu ⁺ cells before, during or after recovery to the Glu ⁺ phenotype.

In one embodiment PTS ⁻ / Glu ⁺ cells may overexpress transketolase encoded by tkt A or tkt B in accordance with the present invention. Transketolase is a pentose phosphorylation pathway enzyme that catalyzes two separate reactions, each producing E4P as a product (see FIG. 1). The tkt A gene is introduced by the introduction of a nucleic acid sequence encoding a transketolase . Amplification (overexpression) can result in increased intracellular concentration of aromatic precursor E4P (US Pat. No. 5,168,056).

In another embodiment, one or more genes encoding a DAHP synthase ( aro G, aro F and aro H) can be introduced or amplified into PTS ⁻ / Glu ⁺ cells in accordance with the present invention. Increased expression of both E4P and DAHP synthase can lead to a significant increase in carbon being passed to the directional pathway compared to strains with only increased DAHP synthase activity.

Thus, in one embodiment, the present invention relates to a host cell that, in addition to a host cell that conserves PEP through inactivation of PTS (PTS ⁻ ), causes a spike in carbon flow due to amplification of transketolase.

It should be borne in mind that when the host cell is cultured in conditions that result in an increase in carbon flow into the directional pathway, it may be necessary to identify and overcome the rate determining step in the pathway. The methodology is useful to those skilled in the art, for example, in US patents. Nos. See 5,168,056 and 5,776,736.

As an example, in the following transition:

For example, under conditions that result in a spike in carbon flow into the pathway of PTS ⁻ / Glu ⁺ and Tkt overexpressing strains, the activity level of DHQ synthase is insufficient to consume as fast as the rate of DAHP formation. As a result of this natural rate determination step in aro B, DAHP is accumulated and secreted into the culture supernatant. This allows the accumulation of DAHP to be used as a method of identifying increased intracellular PEP levels resulting from PTS ⁻ / Glu ⁺ strains.

In addition to increasing the carbon flow through the aromatic pathway, the following genes according to the invention can be overexpressed in PTS ⁻ / Glu ⁺ cells: pps encoding PEP synthase in E. coli (see US Pat. No. 5,985,617). ) And tal A (lida et al. (1993) J. Bacterial. 175: 5375-5383) encoding transaldolase. Moreover, such general aromatic pathways (eg, DAHP synthase (aroF, aroG, aroH), DHQ synthase (aroB), DHQ dehydratase (aroD), schimate dehydrogenase (aroE), chelated kinase (aroL, aroK)) ), Any gene encoding an enzyme that catalyzes reactions in EPSP synthase (aroA) and corismate synthase (aroC)) can be amplified in PTS ⁻ / Glu ⁺ cells included in the present invention.

It will be readily apparent to those skilled in the art that various different genes may be overexpressed depending on the desired product.

In one embodiment, when the desired product koriseu formate, PTS ^- / Glu ⁺ cells the enzyme DAHP synthase, DHQ synthase, according to the present invention; DHQ dehydratase; Shammate dehydrogenase; Schimate kinases; Any one gene in the directional pathway can be overexpressed, including genes encoding EPSP synthase and corismate synthase.

In one embodiment, if the desired product is tryptophan, the enzymes tryptophan synthase (trpA and trpB), phosphoribosyl anthranilate isomerase-indoleglycerol phosphate synthase (trpC), anthranilate phosphoribosyl transferase Any genes can be amplified in the tryptophan-specific region of the directional pathway, including genes encoding (trpD) and anthranilate synthetase (trpE). In another embodiment, the gene encoding tryptophanase (tnaA) may be deficient.

In another embodiment, PTS ⁻ / Glu ⁺ cells may be genetically designed to overexpress pyk according to the present invention if the desired product is pyruvate. The gene encodes pyruvate kinase. If the compound of interest is oxalacetic acid, PTS ⁻ / Glu ⁺ cells can be genetically designed to overexpress ppc encoding PEP carboxylase according to the invention (EC 4.1.1.31).

If the compound of interest is catechol, PTS ⁻ / Glu ⁺ cells may be further transformed with DNA encoding one or more of the following enzyme (s) according to the invention: DAHP synthase (aroF, aroG, aroH) ); 3-dihydroquinate (DHQ) synthase (aroB); Transketolase (tktA or tktB); 3-dihydroscimate (DHS) dehydratase (aroZ) or protocatechuate (PCA) decarboxylase (aroY) (see US Pat. Nos. 5,272,073 and 5,629,181).

Also, for example, if the desired product is adipic acid, one or more of the following enzyme (s) can be overexpressed according to the invention in PTS ⁻ / Glu ⁺ cells (by amplification of the corresponding genes): 3- Dehydroscimate (DHS) dehydratase (aroZ); Protocatechuate (PCA) decarboxylase (aroY) or catechol 1,2-deoxygenase (catA); And, optionally, transketolase (tktA or tktB); DAHP synthase (aroF, aroG, aroH) or DHQ synthase (aroB). (See US Patent 5,487,987).

If the desired product indigo, PTS ^- / Glu ⁺ cells may be transformed with more with the DNA to the tryptophan synthase beta sub-DNA and aromatic encoding a polypeptide analog of unit-deoxy encoding a recombinase according to the present invention. (See US Patent 5,374,543).

Thus, to preserve the PEP material for PEP and PEP precursor command metabolic pathways, for example, an aromatic amino acid route, glycolytic, TCA circuit and PTS that results of increasing the carbon flow into the pentose phosphorylation path ^- / Glu ⁺ In providing strains, the present inventors have provided host systems that can be used to enhance the production of the desired compounds as compared to the production of the same compounds in corresponding PTS host cells.

G. Cell Culture and Fermentation

Methods suitable for the maintenance and growth of bacterial cells are well known and are described in MANUAL OF METHODS OF GENERAL BACTERIOLOGY, edited by P. Gerhardt et al., American Society for Microbiology, Washington DC (1981) and T.D. Brock in BIOTECHNOLOGY: A TEXT B00K OF INDUSTRIAL MICROBIOLOGY, 2nd ed, (1989) Sinauer Associates, Sunderland, MA.

Cell Preculture

Typically cell cultures are grown at 25-32 ° C., and preferably at 28-29 ° C. in a suitable medium. Typical growth media used in the present invention are generally, but are not limited to, commercially prepared media, such as Luria Bertani (LB) liquid media, but also dextrose (SD) liquid media or yeast media (YM) liquid media. . These are available, for example, from GIBCO / BRL (Gaithersburg, MD). Other limited or synthetic growth media may be used and suitable media for the growth of particular bacterial microorganisms will be known to those skilled in the art of microbial or fermentation science. Preferred suitable pH ranges for the fermentation are between pH 5 and pH 8. The preferred range for seed flasks is pH 7 to pH 7.5, and the preferred range for reaction vessels is pH 5 to pH 6. Those skilled in the art of fermentation microorganisms will appreciate that many factors affecting the fermentation process should be optimized and controlled to maximize ascorbic acid intermediate production. Many of these factors, such as pH, carbon source concentration and dissolved oxygen concentration, can affect enzymatic processes depending on the cell type used for the production of ascorbic acid intermediates.

The production of the desired product can be carried out in a fermentative environment, ie in vivo or non-fermentative, ie in vitro environment; Or in a combined in vivo / in vitro environment. The fermentation or bioreactor may be carried out in a batch process or a continuous process.

Fermentation medium

In the present invention, the fermentation medium is a monosaccharide such as glucose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose, and an unrefined mixture from regenerated feedstocks, for example cheese whey permeate, cornsteep liquor), sugar beet molasses, and malt. In addition, the carbon substrate may also be one carbon substrate, such as carbon. It may be contemplated that the carbon source used in the present invention may include a wide variety of carbons containing substrates, but will be limited only by the choice of organism, but preferred carbon substrates may be selected from glucose and / or fructose and mixtures thereof. Include. By using a mixture of glucose and fructose in combination with the altered genome described elsewhere in this application, separation of the oxidative pathway from metabolic pathways can be achieved even while using fructose to meet the metabolic requirements of the host cell. The use of glucose in improved yields and the conversion to the desired ascorbic acid intermediates are possible.

All of the above mentioned carbon substrates are believed to be suitable for the present invention, but preferred are carbohydrate glucose, fructose or sucrose. The concentration of the carbon substrate is from about 55% to about 75% by weight / weight. Preferably, the concentration is about 60 to about 70% by weight / weight. The inventors most preferably used 60% or 67% glucose.

In addition to the appropriate carbon source, the fermentation medium should contain minerals, salts, vitamins, cofactors and buffers suitable for the growth or cultivation and the promotion of the enzymatic pathways necessary for the production of ascorbic acid intermediates.

Batch and Continuous Fermentation

This procedure uses batch, infusion-batch or continuous fermentation methods as culture systems. Such methods are well known in the art and examples can be found in Brock, supra. Classical batch fermentation is a closed system in which the composition of the medium is defined at the start of the fermentation so as not to undergo artificial changes during fermentation. Thus, at the beginning of fermentation, the medium is inoculated with the desired organism or organisms so that the fermentation takes place without adding anything to the system. Typically, however, "batch" fermentation is a batch involving the addition of a carbon source, and adjustment of factors such as pH and oxygen concentration is often attempted. In a batch system the metabolite and biomass composition of the system continues to change until the time when the fermentation is stopped. In a batch culture, cells are relieved to a high growth logarithmic phase through a stationary retardation phase and finally to a stationary stationary growth rate or to a stationary stop. When untreated, the stationary cells will eventually die. Cells in the logarithmic phase generally result in a surge in the production of end products or intermediates.

A change in the standard batch system is an injection-batch system. Feed-batch fermentation procedures are also suitable for the present invention and include typical batch systems except that the substrate is added incrementally with the fermentation progress. Injection-batch systems are useful when metabolite inhibition is likely to interfere with the metabolism of cells and where it is desirable to have a limited amount of substrate in the medium. Determination of the actual substrate concentration in an injection-batch system is difficult and therefore estimated on the basis of changes in the partial pressure of waste gases such as pH, dissolved oxygen and CO ₂ that can be measured.

Although batch or inject-batch methods are preferred in the present invention, continuous fermentation methods may also be used. Continuous fermentation is an open system in which the restriction fermentation medium is continuously added to the bioreactor and the same amount of control medium is removed simultaneously during the process. Continuous fermentation generally maintains the culture at a constant high density at which cells are primarily in logarithmic growth. Continuous culture allows for the adjustment of one factor or any number of factors that affect cell growth or end product concentration. For example, one method would be to keep the limiting nutrients, eg, carbon source or nitrogen levels, at a fixed rate and keep all other parameters in moderation. In other systems, many factors affecting growth may vary continuously, even while the cell concentration, measured as the degree of turbidity of the medium, remains constant. The continuous system strives to maintain steady state growth conditions, so the loss of cells due to media depletion must be balanced against the rate of cell growth in the fermentation. Techniques for maximizing product formation rates, as well as methods for adjusting nutrients and growth factors in a continuous fermentation process are well known in the field of industrial microbiology, and various methods are described in detail by Brock, supra.

The above manner and method of carrying out the invention may be more fully understood by those skilled in the art with reference to the following examples, which embodiments in any way limit the scope of the invention or the claims directed thereto. It was not intended to. All references and patent publications mentioned herein are hereby incorporated by reference.

Experiment

Example 1-PTS ^- E. coli  Construction of the strain

A). lox P-CAT- lox Construction of the P-trc Cassette, Plasmid pTrcM42

Linker DNA was obtained from plasmid pTrc99a (Pharmacia) by cleavage with restriction enzymes HindIII and NcoI as directed by the supplier (New England Biolabs). After purification, the cleaved DNA terminus was determined by Sambrook et al. And T4DNA polymerase as described above. The resulting flat terminal, linear DNA was circulated according to standard protocols and transformed into E. coli TOP-10 competent cells (Invitrgen, Carlsbad CA). Cells were placed in a Luria-agar (LA) plate containing 50 micrograms / ml carbenicillin (5 g / L yeast extract; 10 g / L tryptone, and 10 g / L NaCl + 2% agar). LB medium). After 16 hours of incubation at 37 ° C., 4 colonies were selected for further analysis. Purified plasmid DNA was obtained from the colonies and restriction enzyme analysis was performed. It was confirmed that four colonies contained the same plasmid, and that the DNA region between HindIII and NcoI was removed. The obtained plasmid was named pTrc1.

Plasmid pTrc1 contains only one recognition site for restriction enzyme BspM1 located about 120 bp above the -35 region of the trc promoter. The location was selected and used as a cleavable selection marker. pTrc1 was digested with BspM1 enzyme according to the instructions of the supplier (New England Biolabs). Linear pTrc1 was gel-purified using the QIAquick gel extraction kit (QIAGEN), filled with T4 DNA polymerase described in Sambrook, supra, and linked with loxP-CAT-loxP DNA cassette. loxP-CAT-loxP DNA Cassettes Stu1 and Bam Obtained from plasmid pLoxCAT2 (Palmeros et al., (2000) Gene 247: 255-264) digested with H1. The ligation mixture was transformed into E. coli TOP-10 competent cells (Invitrogen) and applied to luria-agar plates containing 50 micrograms / ml carbenicillin and 20 micrograms / ml chloramphenicol. After 16 hours of incubation at 37 ° C., several colonies appeared on the plate. Portions of the colonies were transferred to fresh LB plates containing carbenicillin and coloramphenicol. After plasmid purification and restriction enzyme analysis, two plasmids containing lox P-CAT- lox P- trc with loxP-CAT-loxP cassettes in the same and opposite directions were selected for the trc promoter, resulting in pTrcm41 and pTrcm42. Was set. (Figure 6)

B). gal For P trc  Construction of Promoter Replacement Template (pR6KgalP)

DNA cassettes containing the trc promoter and lac actuators with the upstream loxP-CAT-loxP cassette were amplified by PCR from pTrcm42 using a primer set of GalA / GalP2.

40 bp in which the primer pair was identical for the gal P upstream region were injected at both ends of the PCR product. The amplification was carried out using 30 cycles (1 min at 95 ° C .; 1 min at 55 ° C .; 2 min at 72 ° C.) using Taq polymerase (Roche). The DNA cassette was cloned into Echo pUni / His5 R6K vector (Invitrogen) and transformed into E. coli Pir1 cells. Positive clones were identified by restriction enzyme cleavage to release the fragments. The construct was designated pR6KgalP.

C). glk  For trc  Construction of Promoter Replacement Template (pR6Kglk).

DNA cassettes containing the trc promoter and lac actuators with the upstream loxP-CAT-loxP cassette were amplified by PCR from pTrcm42 using the GlkA / Glk2 primer set.

40 bp of the primer pair homologous to the gal P upstream region were injected at both ends of the PCR product. The amplification was carried out using 30 cycles (1 min at 95 ° C .; 1 min at 55 ° C .; 2 min at 72 ° C.) using Taq polymerase (Roche). The DNA cassette was cloned into Echo pUni / His5 R6K vector (Invitrogen) and transformed into E. coli Pir1 cells. Positive clones were identified as restriction enzymes and the plasmid was designated pR6Kglk.

D). E. coli  Construction of ptsHlcrr Deleting Strain KLpts7

Of the E. coli PTS KLndh8l ^- a (△ ptsHlcrr) strain (KLp23ndh), pts H, l and pts operon crr total kanamycin resistance marker, including: a (. Levy et al, (1990 ) Gene 86 27-33) Obtained by replacement. This was done by P1 vir transduction using phage digest product 2611 NF9pykF :: G m as described in Flores et al., (1996) Nature Biotechnol , 14: 620-623. Was confirmed by applying the agar + 1% of the colonies on ^{glucose-deletion} of the operon is the deletion and regions amplified by PCR with the hybridization in which primer (ptsHF / crrR) upstream or downstream regions, MacConkey (lactose).

The colonies with deletions in the glucose :: PTS system showed a white phenotype on the plate because they no longer use glucose and produce acid. The strain was designated KLpts7.

E). By linear DNA cassette transformation gal Synthetic exogenous of P natural promoter trc  Substitution with Promoter

rTth RNA polymerase (Perkin Elmer), homology to the E. coli gal P upstream region using pR6K- gal P (SEQ ID NO. 1) and primer pair GalA / GalP2 (SEQ ID NO 6 / SEQ ID NO 7) as template A DNA cassette was generated comprising loxP-CAT-loxP-ptrc with 40 bp of contiguous DNA on both ends. The PCR product is described in Datsenko and Wanner (2000), Proc. Natl. Acad. Sci. USA 97: 6640-6645 was transformed into electro-competent KLpts7 cells comprising pKD46 for integration using the lambda Red system as described. Integration of this cassette produced strain KLpts :: gal P- trc by replacing the regulatory region with a loxP-CAT-loxP-ptrc cassette from 36-183 bp upstream of the gal P ATG start codon (see GenBank Accession U28377).

Colonies were selected on LA plates containing 10 μg / ml chloramphenicol. The integration was amplified by primer pair GalA / GalP2 (SEQ ID NO: 6 / SEQ ID NO: 7) (amplifying the integration site to yield 1.4 kb product) and primer pair GalB1 / GalC11 (integration site including upstream and downstream regions) 2.1 kb product) to confirm by PCR analysis.

PCR parameters were 1 min at 95 ° C. using Taq DNA polymerase or rTth polymerase according to the manufacturer's suggestion; 1 minute at 55 ° C; It was 2 cycles and 30 cycles at 72 degreeC. This strain, the P- gal :: KLpts trc MacConkey agar (lactose ^-) was applied onto a 1% glucose. The colonies showed a cloudy red phenotype compared to KLpts7, indicating that the former strain can make acid from glucose, whereas the latter strain (parent) does not. This confirmed the expression of gal P and that GalP ingested glucose. The promoter region was sequenced to confirm the presence of the promoter. The chloramphenicol markers are described in Palmeros et al. (2000) Gene 247: 255-264] using Cre recombinase as described, and removal by PCR using primer set GalB1 / GalC11 (SEQ ID NO. 10 / SEQ ID NO. 11). Confirmed. The resulting strain was designated KLgalP-ptrc.

F). Synthetic Exogenous of Natural Glucokinase Promoter by Linear DNA Cassette Transformation trc  Replacement to promoter (KLGG)

A DNA cassette comprising loxP-CAT-loxP-ptrc having 40 bp of contiguous DNA homologous to the glk upstream region was prepared as described for the gal P DNA cassette of Example 1E above. The primer set used was GlkA / Glk2 (SEQ ID NO: 149-189 upstream of the glk ATG to the 5 'end and contiguous DNA with the contiguous DNA added to the 3' end from the glk ATG up to 37 bp upstream of the ATG ( glk accession number AE000327). No. 8 and SEQ ID NO. 9). The template used for the PCR amplification was pR6Kglk with rTth polymerase (Perkin Elmer). The glk - trc DNA cassette was transformed into electro-competent KLgalP- trc cells comprising the pKD46 plasmid as described in Datsenko and Wanner (2000) supra. Positive clones were selected on LA agar containing 10 μg / ml of chloramphenicol. The integration of the cassette was confirmed as PCR using the PCR program as described in the construction of primer sets GlkB1 / GlkC11 KLgalP- and trc. GlkB1 the forward primer was to combine, starting from 700 bp upstream of the ATG glk, GlkC11 were combined, starting at 500 bp downstream of the ATG start codon glk.

Colonies are MacConkey agar (lactose ^-) was applied onto a 1% glucose. Colonies were exhibited a deep red, which indicates that the conversion of glucose acid increased compared to the parent (KLgalP- trc). The chloramphenicol marker was removed using Cre recombinase as described in Palmeros et al., Supra, and removal was confirmed by PCR using primer set GlkB1 / GlkC11 to obtain 1.3 kb product. The resulting strain was designated KLGG.

G). Construction of PEP-Independent Glucose Transport System from ptrc Cloned into pACYC177 (pMCGG)

From the trc promoter an appropriate copy number of plasmid to express gal P and glk was constructed. 3040 bp AccI fragments containing gal P and glk respectively under the control of the trc promoter were isolated from plasmid pVHGalPglk11 (FIG. 13). Plasmid pVHGalPglk11 is a low copy number plasmid derived from the pCL1920 vector (Lerner et al. (1990) NAR 18: 4631) with resistance to spectinomycin antibiotics under the control of the trc promoter and the gal P and glk genes from E. coli to be. The nucleotide sequence (SEQ ID NO: 25) of the 3040 bp DNA fragment obtained by AccI cleavage is described in Figures 14A-E. The ends were filled using standard methods (Sambrook et al., Supra). The flat terminal fragment was cloned into the ClaI site of pACYC177 (New England Biolabs) to inactivate the kanamycin resistance gene. Colonies were screened for carbenicillin (100 microgram ml) phase growth and lack of kanamycin (10 μg / ml) phase growth. Isolation of plasmid DNA from positive colonies by standard methods, using XbaI cleavage of the presence of the desired fragment only once in the cloned fragment, separately by restriction digestion with BamHI having two recognition sites in the plasmid Confirmed. This allowed us to determine the arrangement of the injected fragments. The plasmid was designated pMCGG.

H). Build a pSYCO Construct

The use of the PTS ⁻ / Glu ⁺ strain (Example 1E-G) to convert carbon from product to glucose was tested with a plasmid carrying a gene encoding an enzyme that performs the conversion of DHAP to 1,3 propanediol. . The pSYCO construct is a gene (dart1 and gpp2) for the conversion of DHAP (dihydroxyacetone-P) to glycerol from Saccharomyces cerevisiae (hereinafter referred to as the glycerol pathway) followed by Klebsiella ) glycerol and the La (hereinafter referred to 1,3-propanediol route to the 1,3-propanediol from a) a plasmid carrying the gene (dha B1-3, dha X, orf W, X, and Y) for switching based pSC101 (Stratagene). The pSYCO constructs used in the present examples were pSYCO101, 103 and 106, with reference to FIGS. 8 and 9 showing the nucleotide sequence and plasmid map of pSYCO101, respectively. The pSYCO103 construct is identical to pSYCO101 except for the DNA region comprising the glycerol pathway and two EcoR1 recognition sites that are oriented opposite to the direction of pSYCO101. The pSYCO106 construct is identical to pSYCO103 except that the non-coding plasmid DNA 126 bp and 10589-11145 bp were removed between the EcoR1 sites as indicated in FIG. 9. The plasmids for the experiments described herein were functionally equivalent.

Example 2 Encoding Galactose Permease from a Chromosome of a Strain Deficient in a PET-PTS System for Glucose Intake gal Constitutive Expression of P

Preparation of glycerol and 1,3-propanediol in PTS ⁻ / Glu ⁺ E. coli strains with Glu + phenotype was determined. To prepare KLpts7 / pMCGG, the PTS ⁻ / Glu ⁻ strain (KLpts7) was transformed with pMCGG (Example 1G) by standard methods (Sambrook et al. Supra) and produced KlgalP-ptrc (Example 1E). The PTS ⁻ / Glu ⁺ E. coli strain was obtained by chromosomal integration of the trc promoter to replace the endogenous native gal P promoter in KLpts7. Both KLpts7 / pMCGG and KlgalP-ptrc were transformed by standard methods (Sambrook et al., Supra) with plasmids with pathways for glycerol and 1,3-propanediol production. The production of the cell population, glycerol and 1,3-propanediol was tested in fermentation.

Standard fermentation was performed as follows: 500 ml seed flasks were grown at 35 ° C. in standard 2YT medium (Sambrook et al., Supra) for 4-6 hours shaking at 200 rpm. K ₂ HPO ₄ (13.6) using the seed culture; _{KH 2 PO 4 (13.6):} MgSO 4 .7H 2 0 (2); TN 2 medium consisting of citric acid monohydrate (2), ferric ammonium citrate (0.3), (NH ₄ ) ₂ SO ₄ (3.2) yeast extract (5) trace element solution (1 ml) and pH: adjusted to 6.8 A 14 L fermentor operated in glucose transient conditions at pH 6.8, 35 ° C. for 60 hours in (g / L) was incubated.

The trace element solution (g / L) citric acid is _{.H 2 0 (4.0), MnSO} 4 .H 2 0 (3.0), NaCl (1.0), FeSO 4 .7H 2 O (0.10), CoCL 2 .6H 2 0 (0.10), ZnSO ₄ .7H may contain _{2 O (0.10), CuSO 4} .5H 2 O (0.01), H 3 BO 3 (0.01) and _{Na 3 MoO 4 .2H 2 O (} 0.01).

The fermentation was analyzed for cell density by measuring the absorbance of the culture at 600 nM in the absorber, and glycerol and 1,3-propanediol concentrations were determined using HPLC.

Isolation and Identification of 1,3-propanediol:

Conversion of glucose to glycerol and 1,3-propanediol was monitored by HPLC. Analyzes were performed using standard techniques and materials familiar to those skilled in the art of chromatography. One suitable method was the use of a Waters Alliance HPLC system using RI sensing. Aminex equipped with a cationic H refill cartridge precolumn (4.6 mm x 30 mm, Biorad, Hercules, CA) using 5 mM H ₂ SO ₄ as mobile phase at a temperature adjusted to 50 ° C. at a flow rate of 0.4 ml / min. HPX87H column (7.8 mm x 300 mm, BioRad, Hercules, CA) was injected. The system was adjusted weekly to known concentration standards. Typically, the retention times of glucose, glycerol, 1,3-propanediol and acetate were 12.7 minutes, 19.0 minutes, 25.2 minutes and 21.5 minutes, respectively.

In this example, two systems were compared. In the first system, the trc promoter was integrated into the gal P target site (see gal P position-see Example 1E), which causes the E. coli strain to produce glucokinase under natural control of glk , and in the second system the trc promoter Was incorporated into both the gal P target position and the glk target position (see glk position—see Example 1F). 10 and 11 illustrate fermentation results for KLpts7 / pMCGG and KLgalP-ptrc transformed with pSYCO101 and pSYCO103, respectively.

The plasmid encoding the glucose transport system in KLpts7 / pMCGG allowed the strain to grow to high cell density (FIG. 10A) and to produce glycerol and 1,3-propanediol (FIG. 10B). However, the amount of glycerol and 1,3-propanediol produced for this cell population was about 7 g / L of 1,3-propanediol for an OD ₆₀₀ of 194. In contrast, as shown in FIG. 11, KlgalP-ptrc produced much less cell populations and more product 1,3-propanediol about 61 g / L and 36 g / L glycerol (FIG. 11B) in the fermentation (FIG. 11B). 11A).

By constitutively expressing gal P on the chromosome from the trc promoter, the carbon flow from glucose increased towards the desired product, glycerol and 1,3-propanediol, rather than toward the pathway that produces the cell population.

Example 3-PTS ^-  From the chromosome of the strain gal P and glk  Constitutive expression of

The PTS ⁻ / Glu ⁻ strain, KLpts7, was made Glu ⁺ by incorporating the trc promoter into the gal P and glk target sites, resulting in strain KLGG, see Example 1 above. The strain was transformed by standard method using pSYCO101. The production of the cell population, glycerol and 1,3-propanediol was tested in standard fermentation (see Example 2).

As described in FIGS. 11 and 12, KLGG grew faster when compared to Klgal / P-trc (at temperature 33.9, KLGG exhibited an OD ₆₀₀ value of 24.7, whereas OD ₆₀₀ of KlgalP was 19.6). KLGG produced 70 g / L of 1,3-propanediol compared to 61 g / L produced by strain KlgalP-trc. In addition, the maximum concentration was reached earlier in KLGG fermentation (56.8 hours compared to 62.7 hours for KlgalP). Thus, constitutive expression of gal P and glk by chromosomal integration of the trc promoter produced more 1,3-propanediol in a shorter fermentation time than constitutive expression of gal P alone.

Example 4- E. coli  Rapidly Growing PTS ^- / Glu ⁺  Strain Selection and Analysis

Shake flask experiments for KLGG at 24 and 48 hours as shown in Table 2 below show long term retardation during growth demonstrated in fermentation studies of strain KLGG (Example 3 above) during the production of glycerol and 1,3-propanediol. Could be repeated in.

Fast growing strain KLGG was selected by culturing KLGG in fermentor at TM2 and glucose excess conditions at 35 ° C., pH 6.8 to reduce fermentation time. Cells were harvested in the early log phase and applied for colonies isolated on L agar. Isolated colonies were screened to find variants that produce glycerol and 1,3-propanediol upon transformation with pSYCO at a concentration equivalent to KLGG. After confirming the strain, it was determined as FMP. FMP showed the same ability as KLGG, but achieved the same ability within 16 hours of shake flask growth compared to 48 hours for KLGG (FIG. 2).

Shake flask experiments were performed in TM2 with spectinomycin and 2 mg / liter B ₁₂ at a concentration of 2% glucose + 50 micrograms / ml. Overnight cultures of the strain with the pSYCO plasmid were incubated in LB + spectinomycin (50 micrograms / ml) shaking at 200 rpm at 37 ° C. The shaking flasks were inoculated into the 200 microliter overnight culture (10 mls culture in a 250 ml baffle flask) and incubated with shaking at 34 ° C. at 200 rpm. Cell density (OD ₆₀₀ ) for the culture was analyzed and glucose consumption and production of glycerol and 1,3-propanediol were analyzed by HPLC (see Example 2).

Analysis of fast growing strains :: Glucokinase activity, glk mRNA and relative levels of the genes and promoter sequences for the FMP strain were analyzed. As shown in Table 3, the glucokinase activity of FMP was increased three times compared to the activity of KLGG, from 0.08 units to 0.22 units. This suggests an increase in the amount of mutations or existing enzymes in the coding region leading to more active enzymes.

To test expression levels, relative levels of glk mRNA were determined and will be referenced to Tables 4A and 4B. Using a light cycler, results were obtained in the form of crossover times. The shorter the crossover time, the more mRNA was present. Crossover times for gal P were equivalent in KLGG and FMP, indicating similar levels of mRNA in both strains. FMP crossing time of my glk is no shorter than that of KLGG (each about 18.47 to 15.7), the ratio of the average crossing time was 1.18 (KLGG- glk: FMP- glk), which is present in the FMP more glk mRNA strain To indicate. Samples were tested twice (1 or 2) and an average (Avg.) Was taken. The mean was used to determine the ratio ( glk -K / glk -F represent KLGG glk and FMP glk , respectively).

Sequencing was performed on the KLGG and FMP glk genes, and the trc promoter. No mutations were found on the glk coding sequence of FMP. The sequence of the trc promoter was determined by amplification by PCR from the chromosomes of KGLL and FMP using glkB1 / glkBC11 primers. Sequencing was also performed using primer TrcF (SEQ ID NO.14).

Monomutation of G to A was identified as indicated below in the lac effector of the trc promoter in FMP.

Such mutations have been described previously as one of the O ^C effector constitutive mutations that increase the expression of linked genes and decrease the affinity of the effector to lac inhibitors (THE OPERON, Miller, JH and Reznikoff, WS eds. , 1980, p190-192 and references therein). This effectively increases the transcription of glk from the promoter, which has been demonstrated through an increase in enzyme activity. The variant strain grows faster because there is more glucokinase that phosphorylates incoming glucose and more G-6-Ps will be transported to central metabolism.

Assays for Glucokinase Under the Following Conditions

100 mM phosphate buffer pH 7.2, 5 mM MgCl ₂ , 500 mM NADP, 5 mM ATP, 2 units of glucose-6-phosphate dehydrogenase. This assay detects the conversion of glucose and ATP by monitoring the appearance of NADPH ₂ in the formula:

Glucose + ATP → glucose-6-phosphate + ADP

Glucose-6-phosphate + NADP + 2H → Glucono-1,5-lactone-6-phosphate + NADPH ₂

Light cycler determination of relative levels of gal P, glk and 16S rRNA gene ( rrs H) mRNA as a control in shake flask experiments- incubating the strain KLGG and FMP to 20 OD ₆₀₀ in 10 ml + 2% glucose of TM2 It was. The culture was poured directly into liquid nitrogen in 50 ml conical tubes and RNA was purified as described below. The following primers were used:

The light cycler reaction was performed according to the manufacturer's protocol using a Lightcycler RNA Amplication Kit SYBR Green I (Roche) adjusted to 10 μl reactant. A total of 500 ng of RNA was used per reaction. The program used was: a target temperature of 55 ° C .; Incubation time was 10 minutes and the temperature transition rate was 20 ° C./sec.

The RNA isolation method comprises culturing in a shake flask under appropriate conditions as specified in Example 4 above and harvesting by pipetting 7-10 ml directly into liquid nitrogen in a 50 ml conical tube. The samples were frozen at -70 ° C until ready for use. In general, standard methods were used with the following initial adjustments for RNA isolation: 50 ml tubes of frozen samples were placed in a dry ice bucket; 15 ml of phenol: chloroform (1: 1) and 1.5 ml of 3 M NaOAc (pH 4.8) were each added to a 50 ml tube; A small amount of frozen sample (about 500-2000 μl broth) was added to the pre-chilled coffee grinder (with dry ice); Additional dry ice (2 or 3 small pieces) was added to the coffee grinder and the sample was ground for at least 1 minute; Tapping the grinder to obtain all the material in the grinder cap and separating the cap containing the frozen ground cell broth and residual dry ice; An equal amount of 2X RNA extract buffer was quickly pipetted into the cap; The frozen material was stirred into the suspension using a disposable sterile loop and then placed in a conical tube containing 15 ml phenol: chloroform / NaOAc, mixed and placed on ice. Subsequently, standard methods known in the art were performed (Sambrook et al., Supra).

Claims (48)

A method for increasing the carbon flow into the pathway of the bacterial host cells: (PTS phosphotransferase system transport) the PTS was originally available ^{- -} / Glu: phosphate can transport the enzyme to a carbohydrate transport, comprising transport system a) PTS ⁻ / Glu ⁻ host cells of glucose assimilation protein selected from galactosidase permease obtained from E. coli or glucose transporters having at least 80% sequence homology therewith. Nucleic acid encoding a glucose assimilation protein in PTS ⁻ / Glu ⁻ host cells by transformation with a DNA construct comprising a DNA flanking sequence corresponding to an upstream (5 ′) region and a non-host cell promoter or a modified endogenous promoter Changing an endogenous chromosome regulatory region operatively linked to; b) restoring a Glu ⁺ phenotype by incorporation of said DNA construct; And c) culturing the transformed host cell under suitable culture conditions, Wherein the carbon uptake into the metabolic pathway of the transformed host cell is increased relative to the carbon uptake into the same metabolic pathway in corresponding PTS bacterial host cells cultured under essentially the same culture conditions. delete delete The method of claim 1, wherein the glucose assimilation protein is a glucose transporter. delete The method of claim 1, wherein the glucose assimilation protein is a phosphorylated protein. 7. The method of claim 6, wherein said phosphorylated protein is glucokinase. According to claim 1, PTS ^- / Glu ^- a host cell glucosidase to claim transfected with 2 DNA constructs comprising a DNA contiguous sequence and the promoter corresponding to the upstream 5 'region of the kinase PTS ^- / Glu ^- in the host cell Changing the endogenous chromosomal regulatory region operably linked to a nucleic acid encoding a glucokinase. Method according, to which the bacterial host cell is a Escherichia coli (E. coli) cells, Bacillus (Bacillus) cell and the panto O (Pantoea) emitter unit selected for the group consisting of cells according to claim 1. The method of claim 1, wherein said PTS ⁻ / Glu ⁻ host cells are obtained from PTS cells by deletion of one or more genes selected from the group consisting of pts I, pts H and crr . The step of transforming / Glu ⁺ host ^cells of claim trans Kane Tortola azepin trans aldolase and phosphorylation play pyruvate synthase PTS to a polynucleotide encoding a protein selected from the group consisting in the 1 How to further include. To claim 1 in, DAHP synthase, DHQ synthase, DHQ dehydratase, polynucleotide to encoding at least one enzyme selected from the group consisting of formate dehydrogenase, to mate kinase EPSP synthase and koriseu mate synthase in the PTS ^- / Glu ⁺ method further comprising transforming the host cell. A transformed bacterial cell obtained by the method of claim 1. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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